Freshwater mussels of the order Unionida are a widely distributed taxon that are important in maintaining freshwater ecosystems and are also highly imperiled throughout the world. Monitoring of mussel populations with environmental DNA (eDNA) is an attractive alternative to traditional methods because it is noninvasive and requires less labor and taxonomic knowledge from field personnel. We developed eDNA metabarcoding assays specific to freshwater mussels and tested them at six sites in the Clinch River, located in the southeastern United States. Our objective was to determine the utility of eDNA metabarcoding for future monitoring of mussel populations and restoration efforts in this watershed. Two metabarcoding assays that target the mitochondrial DNA regions of the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit (ND1) genes were developed and tested. Our assays appear to be order specific, amplifying members from the two families found in North America, Unionidae and Margaritiferidae, while not amplifying nontarget fish or other bivalve species. From the field collected samples, our assays together detected 19 species, eight of which are listed as federally endangered. The assays also detected 42%, 58%, and 54% of the species identified by recent quantitative visual mussel surveys at three sampling sites. Increased sampling effort by processing a greater water volume or number of samples will likely increase species detections. These eDNA metabarcoding assays may enable enhanced monitoring of freshwater mussel assemblages and subsequently inform conservation efforts.