The habitat requirements of many native freshwater mussels remain unclear despite their imperiled status and ecological importance. To explore scale-specific habitat associations in the three genera of mussels found in the western United States (Anodonta, Gonidea, and Margaritifera) we used a multiscale random forest modeling approach to assess functional habitat parameters throughout a 55 km segment of the upper Middle Fork John Day River in northeastern Oregon. We characterized mussel occurrence and density with respect to the hierarchical, hydrogeomorphic structure by sampling reaches of varying valley confinement and channel units nested within individual reaches. Each genus exhibited unique longitudinal trends and channel unit-use patterns. In particular, the large-scale longitudinal trends in Margaritifera occurrence were associated with hydrogeomorphic characteristics at the reach and channel unit scale, with Margaritifera densities peaking in narrow valley segments and in riffles and runs. At the scale of the channel unit, all mussel genera responded to variation in physical habitat characteristics, particularly those that indicated more stable parts of the channel. Our results suggest that spatial patterns in freshwater mussels are associated with the hierarchical structuring of the lotic ecosystem and may provide guidance to restoration efforts.