The most common definition of a "Non-stop" scheduled line bus, is the operation between station-stops....meaning where passengers can embark or disembark....highway rest stops or driver change or refueling stops do not count, if you can't board or leave at those stops.
With that definition, beginning in 1957, Greyhound Lines began running the world's longest non-stop trip....from New York City to Chicago via the newly completed chain of toll roads....the Lincoln Tunnel, the New Jersey, Pennsylvania, and Ohio Turnpikes, the Indiana Toll Road, and the Chicago Skyway. It made highway rest stops at around four hour intervals, and it made two driver changes at rest stops, at one it refueled. The relief drivers would come from either Pittsburgh or Cleveland in a Company car to the rest stop, and the relieved driver would bring the car back to the terminal.
The trip from end to end took just sixteen hours aboard a Scenicruiser, matching the time of The Broadway and The Century. Everyone boarding at the end points had to travel the whole distance.
Nowadays, the longest nonstopfrom NYC is to Cleveland.
The fuel range of some buses now is over 1200 miles.