“Bartram’s Bass” Micropterus sp. cf. coosae is endemic to the upper Savannah River basin of the southeastern United States and is threatened by hybridization with invasive Alabama Bass Micropterus henshalli. Bartram’s Bass have been functionally extirpated from reservoirs, and hybrid individuals have been detected in several tributaries. However, the extent of introgression in tributaries is currently unknown. Our objectives were to (1) assess the distribution of Bartram’s Bass, native Largemouth Bass M. salmoides, invasive Alabama Bass, and their hybrids in streams of the upper Savannah River basin and (2) quantify effects of abiotic variables on the distribution of each species. We sampled 154 locations in 2017 and 2018 and assigned genetic identity using hydrolysis probes and microsatellites. We used conditional inference trees to quantify variables affecting the occurrence of each species and hybrids. We observed widespread hybridization across the basin. Pure Bartram’s Bass were collected at 27% (42) of sites, among which only 12 sites contained pure Bartram’s Bass and no other congeners. Thirty sites where pure Bartram’s Bass were collected contained hybrids. In the montane Blue Ridge ecoregion, occurrence of pure Bartram’s Bass was negatively affected by low levels of local-scale developed land cover. In the lower-relief Piedmont ecoregion, pure Bartram’s Bass were positively associated with watershed-scale forest land cover and stream gradient. Distance from a reservoir was positively associated with occurrence of pure Bartram’s Bass in both ecoregions. Pure Bartram’s Bass are likely to occur with high probability in only 16% of nonimpounded stream segments; this represents a conservative estimate, and the true number is likely lower. However, future work accounting for incomplete detection of Bartram’s Bass will help to improve confidence in true extirpations. Conservation efforts may be more successful if implemented on stream segments farther from reservoirs or upstream of dispersal barriers preventing colonization of Alabama Bass.