Here's a list of some popular barbecue restaurants in the Orlando area.
(See my review of Bubbalou's below.)
Blackwater BBQ - Orange Ave (my favorite)
O'Boys - Hwy 50 (had takeout from here and it was good)
Uncle Jones - 436 in Casselberry (a down home place)
Cecil's Texas Style Barbecue - Winter Park (never eaten here)
Cecil's Texas Style Barbecue - Orange Ave (ate there once)
Blackwater BBQ (407-888-2033) on Orange Ave has the best barbecue in Orlando and their results in bbq contests on the Memphis in May circuit speak for themselves. You may have seen them on various Food Network shows the past few years. There's one featuring the Big Pig Jig event in Vienna, Georgia that show's the Blackwater crew quite a bit.
The interior of the restaurant has a rustic design, but it was spotlessly clean on the day we visited. It sets back off the road a little bit and we drove by it the first time without seeing it, but nothing a quick U-turn didn't fix. Here's a map.
Here's a review from Orlando City Beat.