Meltwaters on the surface of glaciers have been identified as hot spots for microbial activity. Records indicate that cyanobacteria and green algae dominate the autotrophic assemblages found in the benthic debris in cryoconite holes. Diatoms are commonly recorded in lentic and lotic ecosystems within polar habitats and, in line with the ubiquity principle for microbial communities, potentially, diatoms should be frequently found in the cryoconite of supraglacial environments. In this study, we cultured debris from cryoconite material collected in Svalbard and Greenland, to promote the growth of diatoms. Diatom generic richness varied between 12 and 17 between sites and was ∼5-fold higher than previously reported. Cryoconite supported aerophytic, halophytic, epipelic and bryophilic diatoms, suggesting multiple origins of colonizing cells. Twenty-seven genera were cultured from material that had been frozen (–20°C) for >1 year, indicating their long-term cryotolerance. The diatom flora composition was similar to that recorded in relatively acidic arctic lakes of low conductivity, and bore similarities at the generic level to those from terrestrial/semi-terrestrial moss communities from both the Arctic and Antarctic. As glaciers retreat, the diatom cells residing in cryoconite have the potential to act as seeding agents for a variety of terrestrial and aquatic habitats in proglacial regions and beyond.