A polyphasic approach that included PCR-dependent and PCR-independent molecular techniques was applied to analyze the prokaryotic community in surface waters of shallow Antarctic lakes. The in situ abundance of different bacterial groups was determined by the fluorescence in situ hybridization, whereas bacterial diversity was investigated by 16S rRNA gene sequencing of bacterial clones and isolates. The different approaches allowed the identification of the significant microbial components of the lake bacterioplanktonic communities, indicating a predominance of Flavobacterium, Pseudomonas, and Polaromonas (up to about 56% of total sequences). These genera also appear to be important in freshwater systems elsewhere in the world. Interestingly, closest blast matches to our sequences were predominantly from polar lakes and ponds, in addition to streams and glaciers, suggesting a bipolar distribution of freshwater lake bacterioplankton. Bacteria that are more traditionally associated with the marine environment were also detected, thus indicating an external input by atmospheric deposition and/or seabird excreta. Finally, a slightly different microbial community occurred in the lake at Inexpressible Island that was characterized by low N : P ratio and very high conductivity value, reinforcing the idea that physicochemical and trophic status may affect the structure and composition of the bacterioplankton assemblages in Antarctic lakes.