Best and most popular are two different things, and it's really going to come down to who you ask. In terms of most popular, it probably has to be the movies since they not only bought older fans back into the mix, but also bought in a whole bunch of new fans who never had any previous experience with Transformers.
As for the best, it really depends on personal taste. For me personally, it will always be the Beast Era. I just loved the story and characterization, even though Beast Machines did kind of make a mess of things. G1 was fine for what it was, at the time, but I really don't think it held up that well. I liked Robots in Disguise and Armada (I still maintain Armada had the best incarnation of Starscream), Energon was a piece of crap, and Cybertron was just okay.
The live-action movies were god awful, and I really hated Animated. I could probably do a full rant on what pissed me off about Animated. Though I could probably sum it up as "most disappointing villains ever" and that would cover about half of it. Seriously, did anyone notice the Decepticons were actually doing better BEFORE Megatron got his body back?
As for Prime... I haven't seen a lot of it. It looks interesting from what I have seen, but I haven't seen enough to overly weigh in on it just yet.