The state of Sphaeriidae bivalve mollusc populations is examined in the context of their role in maintaining the sustainability of aquatic ecosystems in the Prypiat sub-basin. Notably, this sub-basin, as part of the Dnipro basin, has a transboundary nature. Based on the field research conducted between 2019 and 2024, this study analyzes the spatial distribution of 18 species in the family, which serve as vital filter feeders in watercourses of varying ranks. Species that are stably capable of forming monopopulations (S. solidum, S. rivicola, S. nuсleus) have been identified, which indicates their key role in the functioning of ecosystems. It should be emphasized that 12 key identified areas where three to eight species coexist, contributing significantly to the functional stability of aquatic ecosystems. Patterns of species distribution in relation to watercourse size were revealed, with the highest occurrence frequency (60–100%) observed in large rivers. This distribution is attributed to the stability of hydrological regimes and the diversity of biotopes. The cluster analysis of the species composition similarity across water bodies allowed the authors to distinguish five main clusters, reflecting variations in the conditions of the family species existence compared to the Horyn reference river. The research suggests that anthropogenic impacts, including 179 cross structures in rivers, 20 significant point sources of pollution, and substantial changes in 75% of river channels, have led to the typical habitat transformations and potentially resulted in the elimination of certain species. These findings are essential for assessing the ecological status of water bodies, expanding the regional conservation network, and developing scientifically grounded measures to enhance aquatic ecosystem sustainability. Furthermore, the importance of the results is also underscored by the protected status of all species in European countries, contrasted with the lack of a protective strategy for these species in Ukraine.