Colored permafrost thaw ponds (thermokarst ponds), are abundant freshwater ecosystems in the subarctic. The ponds can emit significant levels of greenhouse gases, which are drivers of climate change, but little is known of their microbial communities. The ponds are vertically stratified for most of the year with suboxic to anoxic hypolimnia, suggesting varied habitats despite their shallow depths (1 to 4 m). Pelagic bacterial communities in 4 contrasting ponds near the coast of Hudson Bay, Quebec, Canada, were investigated by cloning and sequencing the 16S rRNA gene from environmental DNA. Surface and bottom water libraries yielded a total of 109 operational taxonomical units (defined as 97 % sequence similarity) from 261 clones. The majority of clones had their closest matches to other environmental clones originating from freshwater environments, especially lakes. Proteobacteria accounted for the majority of sequences (33 %), followed by Bacteroidetes (19%), Actinobacteria (13%), Chlorobi (10%), Verrucomicrobia (8%), and Cyanobacteria (7%). There were large differences in the assemblages inhabiting the different ponds and water layers. Bacteroidetes were predominant at the surface, while Chlorobi were found in bottom waters. Sequences with best matches to known methanotrophic bacteria represented up to 20% of all sequences. Methanotrophs included Methylobacter psychrophilus, Crenothrix polyspora, and Methylocystis parvus as closest matches. Ordination analysis clearly separated surface and bottom waters, where relatively large quantities of organic matter, low light availability, cool temperatures, and suboxic conditions selected for the distinct communities. The separation of surface waters of different ponds showed that bacterial communities could differ between superficially similar environments. This study illustrates the high diversity in bacterial assemblages occurring in ponds separated by a few meters, and thus, the potentially complex response of thaw pond systems to climate change.