Cultivation techniques were used to study the heterotrophic bacterial diversity in two microbial mat samples originating from the littoral zone of two continental Antarctic lakes (Forlidas Pond and Lundström Lake) in the Dufek Massif (within the Pensacola Mountains group of the Transantarctic Mountains) and Shackleton Range, respectively. Nearly 800 isolates were picked after incubation on several growth media at different temperatures. They were grouped using a whole-genome fingerprinting technique, repetitive element palindromic PCR and partial 16S rRNA gene sequencing. Phylogenetic analysis of the complete 16S rRNA gene sequences of 82 representatives showed that the isolates belonged to four major phylogenetic groups: Actinobacteria, Bacteroidetes, Proteobacteria and Firmicutes. A relatively large difference between the samples was apparent. Forlidas Pond is a completely frozen water body underlain by hypersaline brine, with summer thaw forming a slightly saline littoral moat. This was reflected in the bacterial diversity with a dominance of isolates belonging to Firmicutes, whereas isolates from the freshwater Lundström Lake revealed a dominance of Actinobacteria. A total of 42 different genera were recovered, including first records from Antarctica for Albidiferax, Bosea, Curvibacter, Luteimonas, Ornithinibacillus, Pseudoxanthomonas, Sphingopyxis and Spirosoma. Additionally, a considerable number of potential new species and new genera were recovered distributed over different phylogenetic groups. For several species where previously only the type strain was available in cultivation, we report additional strains. Comparison with public databases showed that overall, 72% of the phylotypes are cosmopolitan whereas 23% are currently only known from Antarctica. However, for the Bacteroidetes, the majority of the phylotypes recovered are at present known only from Antarctica and many of these represent previously unknown species.