Are there any rules of thumb for the most comfortable seats on a long distance bus?

One other thing to consider when choosing which side of the bus to sit on is where the sun will be during your trip, based on the time of day and overall direction of travel. Sitting on the sunny side of the bus can be less comfortable in terms of both temperature and glare, so I generally choose to sit on the shady side if possible.

I'd say, sit at least a couple of rows away from the bathroom; and skip the first row behind the driver than the door; they sometimes have less leg room and no view (at least behind the driver).

If you know where you are going, sitting on the curb side or street side can have different views.

(personal experience) For example, when in a bus between Sorrento and Amalfi (Italy), it is more fun sitting on the sea side than on the mountain side

On some mountain routes (I've experienced this in Switzerland) the back of the bus swings out over the edge of the road on bends -- and it's a long way down, so sit near the front if you're afraid of heights. These buses often have the back axle a long way forwards, which makes my next point more significant.

Leverage suggests that you should sit between the axles if you're worried abut motion sickness. If the back axle goes over a bump and you're on the back row, you can be quite a long way behind the axle, increasing the motion due to the bump. Also right at the back the engine vibrations can be more noticeable, again not good if you get travel sick.

The seats can get rather warm if they're directly over the engine (e.g. many UK urban buses, but also on long-distance routes depending on the configuration). This could be good or bad for you depending on whether you feel the cold. On really old or badly maintained buses there may be a smell of hot oil in this area -- again one to avoid if you get sick.

I've traveled extensively by long-distance bus, and can say from experience that the best seats really depend on what you're looking for. Some bus carriers have two levels, some have outlets, most have wifi.

Front vs. Back

If noise is a concern, you're better off closer to the front, as most buses these days have their engine in the back. Also, bathrooms tend to be in back, so unless you predict frequent bathroom visits, you're better off up front for smell. You also generally have the worst view towards the back in a rear-engine bus, because having the engine in back means no rear windows. It doesn't seem to matter much for vibration/bouncing where in the bus you are.

Left vs. Right

I'd pick the side of the bus depending on what you want to see, notably sunrise and sunset. In the northern hemisphere, the sun is always to the south, and regardless of hemisphere it rises in the east and sets in the west. If you're going north and you want to catch the sunrise, that's the right side of the bus. South, left side. North and trying to catch sunset, left side. South, right side. Going east the sun will be a little to your right, going west it'll be to your left. You can see other features the same way. Say you're taking a bus up the west coast and you want a chance to see the ocean. That's west, so going north puts the coast on your left. Of course, if you're planning on sleeping as much of the ride as possible, you might want to be on the opposite side from the sun.

Window vs. Aisle

This is really a matter of personal preference. Window gives you views and usually better access to the outlets, which tend to be along the wall, while aisle gives you a bit more space for your things. If you're particularly social or need to keep an eye on the family, aisle also gives you better access to other people in the bus. In my experience, aisle tends to get more claustrophobic, because you get less window views. Unlike in an airplane though, where the windows are tiny, you'll be able to see out the windows regardless of where you are.

I personally prefer the window, unless I'm sitting at my favorite seat, described below.

Seating Types

A good number of the big bus carriers don't have assigned seating, so you're stuck showing up early and hoping for something good. A few of the big carriers provide reserved seating for another dollar or two, which lets you pick your seat in advance out of a number of numbered, slightly more comfortable seats to the front. A few carriers also have some seats with tables, which is handy if you want to get work done or comfortably seat your family. If it's leg room you're after, you can get seating with your feet by the stairs, leaving more room for yourself and your things. This is often called balcony seating. They'll rarely let you leave things on the stairs themselves though. Note that reserved, balcony or table seating will usually cost from $1 to $3 more per ride, which tends to be well worth it for an hours-long trip.

My personal favorite perk is provided by Megabus, and probably a few other carriers as well: panoramic window views at the front of the upper level. Rather than putting regular windows at the front of the upper level of the bus, someone thought to put a giant window across the entire front, offering amazing views for $5 to $9 more, which for a long ride buys you hours full of entertainment, and the extra space up front. Try finding amusement park tickets for under $10. There's also a sun shield you can pull down over the window if you want to go to sleep.

The real advantage of buying reserved seats is you know what you're going to get ahead of time, and never have to risk smelly bathroom-side seats if you show up at the end of the line.

The Takeaway

You're basically always best off closer to the front, which is why that's where they put most of their reserved seating. Pick your side based on the views and the sun, and how much sleep you're planning on getting. Pick whether you want a window or aisle seat depending on whether views or the ability to get up and move are more important to you, though you'll generally not be doing much getting up and moving on such a bus. I'd always go with reserved seating, because it guarantees you a good seat of your choice rather than leaving it up to chance. Finally, if you have such an opportunity, pick one of the four front seats on top, and you'll love the views. Plus, the panoramic glass means they're all window seats, so you can go with one of the middle two aisle seats for extra space.

Enjoy your trip, and happy travels!

In hot areas, check for ventilation openings/emergency exits in the ceiling, open it, and seat yourself in the aisle one or two seats behind it for some natural ventilation.

The three in-line seats next to the bathroom FTW. Nap time. I'm short and can get comfortable almost anywhere, so your millage may vary.

Most of the journey will be highway miles, at least in the States, and if you're like me you won't be able to sleep sitting up or through any of the stops anyway. So if you can get the couch in the back all to yourself, take it. Because none of the seats are "comfortable".

If you can't, then differ to other answers that mention where you'd want to be relative to the wheel base, not the sunny side, and isle seats.

On a personal note, because you're considering long distance buses, the three and a half day trip I took across the States is something that I will never, ever, repeat. It's on my top ten list of things I'd be willing to criminalize myself to avoid if necessary.

There is one aspect that no one referred. I noticed in my last trip and may be important for some.

Picking the first row of sits assures you that no one reclines the chair towards you. Picking the last, before the back door allows you to recline your chair without bothering anyone.

This might be particularly important for night trips.