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Arctic lakes, ponds and streams contain benthic microbial mats that are dominated by cyanobacteria, and these communities often account for a large proportion of the total ecosystem biomass and productivity. The vertical structure and composition of mats from two different aquatic habitats in the Canadian High Arctic, Ward Hunt Lake and a polar desert stream were analyzed in detail by microscopy techniques. Two distinct layers were identified in each mat: a surface layer with a high density of cells and associated extracellular polymeric substances (EPS), and a less cohesive bottom layer with an accumulation of mineral particles. The matrix formed by the cyanobacterial filaments and EPS produced the complex microstructure of all three mats, and likely favoured different microenvironments where specialized microbial interactions and biomineralization processes could take place. Structural and compositional differences were found among the mats. The lake mat had a surface layer of Dichothrix, and contained abundant particles of calcium carbonate, while Tychonema-like and Tolypothrix-like appeared mainly in the stream mats, along with a higher diversity of Chroococcales. A black microbial mat from one of the stream sites had markedly lower diversity than the other mat types. The observed differences in cyanobacterial composition and physical structure may be related to habitat stability and the availability of liquid water.