The Buffalo River in Tennessee historically contained a rich diversity of freshwater mussels. Sampling efforts in the 1980s documented declines in most of the main channel. Recent collection data indicated recovery in the upper and lower reaches of the river. The objective of this study was to update the current status, distribution, and species composition of the mussel fauna in the main channel and major tributaries through qualitative sampling, and document community structure using quantitative sampling at the most diverse location in the main channel for use in future monitoring efforts. In the qualitative sampling portion of this study, timed searches established catch-per-unit effort (CPUE) at intervals of ∼5 river miles in the main channel and tributaries. We recorded a total of 36 species at 62 sites, including 3 federally protected species: Margaritifera monodonta (Spectaclecase), Pleuronaia dolabelloides (Slabside Pearlymussel), and Theliderma cylindrica (Rabbitsfoot). An additional 3 species being considered for the federal endangered species list were also found extant in the main channel: Obovaria subrotunda (Round Hickorynut), Pleuronaia barnesiana (Tennessee Pigtoe), and Toxolasma lividum (Purple Lilliput). Multiple sites in the upper and lower main-stem were suitable for reintroduction of species. These findings should be considered in future management and conservation efforts.