Freshwater mussels and stream substrate were sampled at 30 locations in the Apalachicola, Chattahoochee, and Flint (ACF) river basins. Although >2100 mussels that included 25 species were observed in our sample of 2661 quadrats, only 5 species were sufficiently abundant to provide reliable estimates of the relationship between mussel presence and substrate composition. Among these 5 species, only Villosa lienosa was associated with substrate composition. Villosa lienosa was most prevalent in well-sorted sediments that contained high proportions of fine particles, but its presence was unrelated to sediment porosity. Because many species of freshwater mussels in these Coastal Plain streams are dispersed and rare, future studies of mussel–habitat associations in the ACF basin should include novel sampling designs and methods that allow rare species to be encountered in greater numbers than those observed in our study.