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Cyanobacteria of biological soil crusts (BSCs) represent an important part of circumpolar and Alpine ecosystems, serve as indicators for ecological condition and climate change, and function as ecosystem engineers by soil stabilization or carbon and nitrogen input. The characterization of cyanobacteria from both polar regions remains extremely important to understand geographic distribution patterns and community compositions. This study is the first of its kind revealing the efficiency of combining denaturing gradient gel electrophoresis (DGGE), light microscopy and culture-based 16S rRNA gene sequencing, applied to polar and Alpine cyanobacteria dominated BSCs. This study aimed to show the living proportion of cyanobacteria as an extension to previously published meta-transcriptome data of the same study sites. Molecular fingerprints showed a distinct clustering of cyanobacterial communities with a close relationship between Arctic and Alpine populations, which differed from those found in Antarctica. Species richness and diversity supported these results, which were also confirmed by microscopic investigations of living cyanobacteria from the BSCs. Isolate-based sequencing corroborated these trends as cold biome clades were assigned, which included a potentially new Arctic clade of Oculatella. Thus, our results contribute to the debate regarding biogeography of cyanobacteria of cold biomes.