The western sand darter Ammocrypta clara, and eastern sand darter Ammocrypta pellucida, are sand-dwelling fishes of conservation concern. Past research has emphasized the importance of studying individual populations of conservation concern, while recent research has revealed the importance of incorporating landscape scale processes that structure habitat mosaics and local populations. We examined habitat use and distributions of western and eastern sand darters in the lower Elk River of West Virginia. At the sandbar habitat use scale, western sand darters were detected in sandbars with greater area, higher proportions of coarse grain sand and faster bottom current velocity, while the eastern sand darter used a wider range of sandbar habitats. The landscape scale analysis revealed that contributing drainage area was an important predictor for both species, while sinuosity, which presumably represents valley type, also contributed to the western sand darter’s habitat suitability. Sandbar quality (area, grain size, and velocity) and fluvial geomorphic variables (drainage area and valley type) are likely key driving factors structuring sand darter distributions in the Elk River. This multiscale study of within-river species distribution and habitat use is unique, given that only a few sympatric populations are known of western and eastern sand darters.