We conducted a riverscape analysis of Rainbow Darters, Etheostoma caeruleum, across their range. We aimed to evaluate population genetic structure and diversity and to identify the possible drivers, contemporary or historical, that underlie the genetic patterns. This study genotyped 508 Rainbow Darters, collected from 67 locations across 23 river basins, at ten DNA microsatellite loci. Genetic clusters were assessed using STRUCTURE, Akaike information criteria (AIC), and discriminant analysis of principal components (DAPC). The STRUCTURE analysis identified two genetic clusters, one comprised of fish collected from more southerly sites and one comprised of fish from more northerly sites. Interestingly, the two clusters predominantly corresponded to sites that were either unglaciated or glaciated during the last glacial maximum (LGM). The AIC and DAPC identified additional structure, including one genetically distinct population in the Meramec River basin and another in the “Driftless” area of the upper Mississippi. Additional genetic clusters also occurred within the glaciated region, which may reflect expansion from different refugia or limited gene flow across river basins since the LGM. Our findings demonstrate the lasting legacy of glacial history on the contemporary genetic structure and diversity of a North American riverine species.