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Cyanobacterial mats are commonly reported as hotspots of microbial diversity across polar environments. These thick, multilayered microbial communities provide a refuge from extreme environmental conditions, with many species able to grow and coexist despite the low allochthonous nutrient inputs. The visibly dominant phototrophic biomass is dependent on internal nutrient recycling by heterotrophic organisms within the mats; however, the specific contribution of heterotrophic protists remains little explored. In this study, mat community diversity was examined along a latitudinal gradient (55-83°N), spanning subarctic taiga, tundra, polar desert, and the High Arctic ice shelves. The prokaryotic and eukaryotic communities were targeted, respectively, by V4 16S ribosomal RNA (rRNA) and V9 18S rRNA gene amplicon high-throughput sequencing. Prokaryotic and eukaryotic richness decreased, in tandem with decreasing temperatures and shorter seasons of light availability, from the subarctic to the High Arctic. Taxonomy-based annotation of the protist community revealed diverse phototrophic, mixotrophic, and heterotrophic genera in all mat communities, with fewer parasitic taxa in High Arctic communities. Co-occurrence network analysis identified greater heterogeneity in eukaryotic than prokaryotic community structure among cyanobacterial mats across the Canadian Arctic. Our findings highlight the sensitivity of microbial eukaryotes to environmental gradients across northern high latitudes., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)