While a majority of Florida lakes are shallow, several deep lakes exist and are thermally stratified (divided into two layers: epilimnion and hypolimnion) during the majority of the year. However, little is known about microbial communities and their functions in each layer of these thermally stratified, subtropical Florida lakes. This study attempts to compare the epilimnion with the hypolimnion of freshwater Florida lakes in terms of both chemical and microbial factors. Water samples were collected from four deep lakes in north (Johnson and Sheelar) and central (Verona and Tulane) Florida at two depths and were analyzed for microbial communities using DNA sequencing. Alpha diversity analysis revealed that microbial diversity was significantly higher (avg~3300OTUs) in the hypolimnion than in the epilimnion (avg ~1400OTUs) An ordination plot revealed that epilimnion microbial community compositions significantly differ from those epilimnion. The effect of lake on microbial communities was also observed as microbial communities in oligotrophic lakes differ from those in meso-and eutrophic lakes. These results suggest that microbial processes that govern biogeochemical cycles in lake differ between epilimnion and hypolimnion of lakes. The data gathered in this study can be applied to understand microbial mediated biogeochemical cycles in subtropical deep lakes during stratification.