Freshwater unionid bivalves play an important role in nutrient cycling and organic matter transportation and provide hard substrates for other benthic animals in freshwater ecosystems. Therefore, the conservation of unionid bivalves is considered essential for maintaining aquatic biodiversity. In this study, the assimilated food sources of six species of freshwater bivalves, Unio douglasiae, Unio douglasiae nipponensis, Unio biwae, Anodonta japonica, Pronodularia japonensis, and Lanceolaria grayana, were investigated using fatty acid trophic markers. The contribution (percentage of total identified fatty acids) of the trophic markers showed a similar tendency in all the bivalve species, even though they were sampled from various habitats. Fatty acid trophic markers of diatoms (20:5n3) and green algae and/or cyanobacteria (18:2n6 18:3n3) were dominant in all bivalve samples, ranging from 4.8% to 10.2% and 7.3% to 10.6%, respectively. Bacterial fatty acids were also detected in large amounts, ranging from 1.7% to 5.4%. In this study, all unionid bivalves contained diatom, green algal/cyanobacterial, and bacterial markers in substantial proportions, and no individual depended on a single food source, stressing the importance of ingesting and assimilating various food types.