The Soda Poppers theme song is a parody of the theme to the 1960s Batman TV series.
The game won the Gamespot Editor's Choice 2006 Award for the category Funniest Game and was elected by IGN as the PC Adventure Game of the Year 2006.
Jesse James's severed hand (which played a key role in Hit the Road) is on a plaque above Sam & Max's closet.
LucasArts was producing a sequel to Sam and Max Hit the Road called Sam and Max: Freelance Police. The game would have been a 3D point and click adventure in the style of the original. When it was canceled in 2004, many members of the development team left LucasArts to help form Telltale Games, the company that eventually ended up developing Sam and Max: Culture Shock.
In Sam and Max's office, there's a cardboard box with the date 03-03-04. If you look at it, Sam will say "That was a particularly gruesome case..." This is the exact date that LucasArts announced the cancellation of Sam and Max: Freelance Police.