The Paint Rock River (PRR) drainage in northern Alabama and southern Tennessee historically supported 58 freshwater mussel species. This study semi-quantitatively examined the mussel assemblage at 42 sites in the Paint Rock mainstem and 5 sites in Estill Fork, a headwater tributary. A total of 1825 live mussels were collected over 78.9 person-hours, with an overall catch per unit effort (CPUE) of 23.1 mussels/person-hour. Forty-one species were collected live and/or fresh dead, including federally protected Epioblasma triquetra (Snuffbox), Fusconaia cor (Shiny Pigtoe), Lampsilis abrupta (Pink Mucket), Lampsilis virescens (Alabama Lampmussel), Pleuronaia dolabelloides (Tennessee Pigtoe), Quadrula cylindrica cylindrica (Rabbitsfoot), and Toxolasma cylindrellus (Pale Lilliput). The river system continues to support a high diversity of mussels (48 species collected in the past 25 years). The survey also identified several sites in the basin suitable for the reintroduction of extirpated species.