Climate change is causing rapid permafrost degradation across Arctic and subarctic regions, resulting in changes in the size, abundance, and structure of thermokarst (thaw) ponds and lakes. The main objectives of this study were to analyze periphytic diatom communities and their affinity to vegetation substrates in thermokarst ecosystems located in the eastern Hudson Bay region and to establish a first inventory of diatom assemblages and the associated littoral vegetation in these systems. Some generalist diatom species, including Tabellaria flocculosa, occupied all ecological niches in the water bodies. In contrast, genera such as Eunotia and Pinnularia were more specialized and generally concentrated on moss substrates. Shoreline vegetation and thermokarst pond/lake littoral morphology (slope) resulted in limnological conditions that differed between sites and ultimately affected diatom community structure. Our results show that both shoreline vegetation and diatom communities are diverse in thermokarst ecosystems, and their species composition depends mostly on site-specific properties (available microhabitats, local pond/lake morphology) rather than limnological conditions that are closely aligned with regional ecoclimatic conditions.