As large, long-lived filter feeders with high clearance rates, unionid mussels are capable of altering nutrient cycling in freshwater food webs. Because the effects of mussel communities on ecosystem processes result largely from their feeding behavior, we need to understand how they sample and process particulate matter. Our objective was to determine whether measuring a suite of biochemical markers in unionid mussels, from different populations of 2 species (Amblema plicata, Actinonaias ligamentina) across a broad geographical range (4 North American rivers), would help identify their primary dietary sources. Stable isotope data were able to differentiate mussels among rivers, and sometimes species. Data on δ¹⁵N revealed that mussels fed across trophic levels but that diet varied with habitat and perhaps species. Although mussels contained the same types of fatty acids regardless of river or species, we nonetheless observed considerable variation in essential fatty acid content across rivers. Essential fatty acid profiles were dominated consistently by arachidonic acid. Source-specific fatty acid biomarkers suggested that bacterial and detrital resources might be as important as algae in many habitats. In summary, biochemical metrics differed more among rivers than between species. This suggests that mussels are either considerably adaptable in their dietary requirements or that they feed nonselectively. Understanding nutritional profiles in mussels will help restore unionid communities, which may lead to the reestablishment of their critical role as nutrient recyclers in freshwater food webs.