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Antarctic lake ecosystems provide a rare opportunity to study the evolution and adaptation of microorganisms to extreme conditions, as well as to discover new species useful for biotechnological applications. Four water samples were collected from various layers of the water column of freshwater Lake Radok in East Antarctica. Two regions (v3-v5 and v4-v8) of the 16S rRNA gene were amplified by PCR and sequenced. Twenty dominant phylotypes were detected representing five bacterial phyla (Actinobacteria, alpha, beta and delta Proteobacteria, Bacteroidetes, Planctomycetes, OD1) and two eukaryotic divisions (stramenopiles and green algae). Of these, 16 phylotypes demonstrated <= 98 % identity to the nearest taxa in GenBank and were therefore classified as new unknown species. Seven phylotypes demonstrated <= 90 % identity and thus remained unidentified. Actinobacteria was the most abundant phylum with 157 clones (41 % of the total number) representing 5 phylotypes. A species complex (3 clades from acI-A subgroup) of Candidatus Planktophila limnetica was prevalent in all layers. Representatives of the OD1 phylum and delta-proteobacteria were discovered by sequencing of the v3-v5 region of 16S rRNA, while Planctomycetes, beta-proteobacteria and mtDNA of stramenopiles were discovered by sequencing of the v4-v8 region. This highlights the necessity of sequencing at least two 16S rRNA gene regions to gain more data on microbial community characterization. In general, despite the uniformity in the physical and chemical properties in the water column, a prominent stratification of microbial groups was observed, at the levels of both divisions and phylotypes.