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Microbial mats exist in freshwater pools atop the Ward Hunt and Markham ice shelves in the Canadian high Arctic. In this study, culture-dependent and culture-independent techniques were used to describe the microbial diversity and activity of these mat communities.Bacterial and archaeal 16S rRNA gene clone libraries were constructed from Markham and Ward Hunt mat samples. Bacterial libraries from both mats had high diversity, though the Markham library appeared more diverse than the Ward Hunt library. Over 95% of sequences in both bacterial libraries, and all isolates from both mats, grouped within the phyla Bacteroidetes, Proteobacteria, and Actinobacteria. Archaeal diversity appeared low in both mats. Only one phylotype, potentially representing a novel Euryarchaeota, was observed in both archaeal libraries. Populations in both communities showed activity at subzero temperatures, with growth of isolates at -5°C and detectable metabolic activity at -10°C, measured by radiorespiration assays of mat microcosms.