Build a smart playlist that has criteria to select songs by "least often played". You have to choose the "Limit to ..." criteria to get this option, at the bottom of the SPL definition. You can also add other criteria of course, such as song rating, date added, etc.
My only criteria is least often played. I have limited to 25 songs. All the other selection fields are empty because I just want the one criteria. Itunes then says there are no songs to match my criteria. Am I missing something?