16S rRNA clone library analysis was used to examine the biodiversity and community structure within the sediments of three hypersaline Antarctic lakes. Compared to sediment of low to moderate salinity Antarctic lakes the species richness of the hypersaline lake sediments was 2–20 times lower. The community of Deep Lake (32% salinity, average sediment temperature −15°C) was made up almost entirely of halophilic Archaea. The sediment communities of two meromictic hypersaline lakes, Organic Lake (20% salinity, −7°C) and Ekho Lake (15% salinity, 15°C) were more complex, containing phylotypes clustering within the Proteobacteria and Cytophagales divisions and with algal chloroplasts. Many phylotypes of these lakes were related to taxa more adapted to marine-like salinity and perhaps derive from bacteria exported into the sediment from the lower salinity surface waters. The Ekho Lake clone library contained several major phylotypes related to the Haloanaerobiales, the growth of which appears to be promoted by the comparatively high in situ temperature of this lake.