| Summary: | We assessed fungal diversity in water and sediment samples obtained from five Arctic lakes in Ny-Ålesund (Svalbard Islands, High Arctic) and five Antarctic lakes on Livingston and Deception Islands (South Shetland Islands), using DNA metabarcoding. A total of 1,639,074 fungal DNA reads were detected and assigned to 5980 ASVs amplicon sequence variants (ASVs), with only 102 (1.7%) that were shared between the two Polar regions. For Arctic lakes, unknown fungal taxa dominated the sequence assemblages, suggesting the dominance of possibly undescribed fungi. The phylum <i>Chytridiomycota</i> was the most represented in the majority of Arctic and Antarctic samples, followed by <i>Rozellomycota</i>, <i>Ascomycota</i>, <i>Basidiomycota</i>, and the less frequent <i>Monoblepharomycota</i>, <i>Aphelidiomycota</i>, <i>Mortierellomycota</i>, <i>Mucoromycota,</i> and <i>Neocallimastigomycota</i>. At the genus level, the most abundant genera included psychrotolerant and cosmopolitan cold-adapted fungi including <i>Alternaria</i>, <i>Cladosporium</i>, <i>Cadophora</i>, <i>Ulvella</i> (Ascomycota), <i>Leucosporidium, Vishniacozyma</i> (<i>Basidiomycota</i>), and <i>Betamyces</i> (<i>Chytridiomycota</i>). The assemblages displayed high diversity and richness. The assigned diversity was composed mainly of taxa recognized as saprophytic fungi, followed by pathogenic and symbiotic fungi.
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