We compared feeding habits and trophic-level relationships of unionid species in a detritus-dominated river and an alga-dominated lake using biochemical analyses, gut contents, and stable-isotope ratios. The δ13C ratios for algae and other food-web components show that all unionids from both the river and the lake used bacterial carbons, not algal carbons, as their main dietary source, in spite of positive selection and concentration of diatoms and green algae from the water column in the gut and mantle cavity. Algae did provide key nutrients such as vitamins A and D and phytosterols that were bioaccumulated in the tissues of all species. The δ15N ratios for the multispecies unionid community in the Huron River indicated some differences in nitrogen enrichment between species, the greatest enrichment being found in Pyganadon grandis. These δ15N ratios indicate that unionids may not always feed as primary consumers or omnivores. Stable-isotope data were critical for delineating diets and trophic-level interactions of this group of filter-feeders. Further refinements in identifying bacterial and picoplankton components of the fine particulate organic matter are needed to complete our understanding of resource partitioning between multispecies unionid populations.