Freshwater mussels (family Unionidae) are among the world's most endangered taxa, with almost 75% of North American taxa classified as a species of concern, threatened, or endangered. Despite the critical importance of comprehensive distributional data for the conservation of unionids and fishes, these data are often lacking because of the labor and resources associated with traditional survey methods. During their larval stage, unionid mussels use various fish species as obligate hosts, making native fish species vital to unionid persistence and an understanding of host distribution similarly important. Here, we utilized an eDNA metabarcoding approach to evaluate patterns of co‐distribution of unionid mussels and fishes along ~362 km of the densely sampled Grand River network as well as the outlets of 19 tributaries along the eastern shore of Lake Michigan, USA. We detected a total of 21 mussel and 40 fish taxa, with distinctive composition of both mussel and fish assemblages across tributaries and differences in fish taxa between sampling periods. Notably, we detected more mussel taxa within the Grand River watershed than at the outlets of all 20 rivers combined. Within the Grand River network, two fish taxa (Pylodictus olivaris and Cyprinella) were found more frequently in areas of high mussel diversity, and three fish taxa more frequently in areas of low mussel diversity (Umbra, Leuciscidae, and Etheostoma). There was little difference between eDNA detections of mussels from samples collected in June versus August, but we detected significantly more fish taxa in August compared to June. Taken together, our findings demonstrate the value of eDNA metabarcoding for evaluating co‐distribution of ecologically connected taxa. The use of eDNA as a tool for determining distributions of mussels and their obligate hosts may facilitate conservation efforts for these imperiled taxa.