A nearly 40-year debate on the origins of carbon supporting animal production in lotic systems has spawned numerous conceptual theories emphasizing the importance of autochthonous carbon, terrestrial carbon, or both (depending on river stage height). Testing theories has been hampered by lack of adequate analytical methods to distinguish in consumer tissue between ultimate autochthonous and allochthonous carbon. Investigators initially relied on assimilation efficiencies of gut contents and later on bulk tissue stable isotope analysis or fatty acid methods. The newest technique in amino acid, compound specific, stable isotope analysis (AA-CSIA), however, enables investigators to link consumers to food sources by tracing essential amino acids from producers to consumers. We used AA-CSIA to evaluate nutrient sources for 5 invertivorous and 6 piscivorous species in 2 hydrogeomorphically contrasting large rivers: the anastomosing Upper Mississippi River (UMR) and the mostly constricted lower Ohio River (LOR). Museum specimens we analyzed isotopically had been collected by other investigators over many decades (UMR: 1900–1969; LOR: 1931–1970). Our results demonstrate that on average algae contributed 58.5% (LOR) to 75.6% (UMR) of fish diets. The next highest estimated contributions of food sources were from C3 terrestrial plants (21.1 and 11.5% for the LOR and UMR, respectively). Moreover, results from 11 individually examined species consistently demonstrated the importance of algae for most fish species in these trophic guilds. Differences among rivers in relative food source availability resulting from contrasting hydrogeomorphic complexity may account for relative proportions of amino acids derived from algae.