Striped bass is the subject of important commercial and sport fisheries in North America. The Roanoke River drainage—especially Smith Mountain Lake, Leesville Lake, and Kerr Reservoir—has popular recreational striped bass fisheries. After construction of five hydroelectric dams, populations became landlocked, declined, and have been supplemented by stocking. A key basis for responsibly augmenting populations is to characterize genetic variation and incorporate the findings into responsible hatchery and stocking practices. Genetic variation at 12 microsatellite DNA loci was evaluated among 837 striped bass representing 16 collections across the native range; populations from rivers in South Carolina, North Carolina, Chesapeake Bay, and Hudson River were screened to provide context for assessing genetic structure within the Roanoke system. Analysis of population genetic differentiation showed landlocked Roanoke River striped bass to be distinctive. Subject to genetic isolation, high M ratios, and relatively low Ne estimates suggest loss of genetic variation, and relatedness analysis showed heightened frequencies of related individuals. These insights into population genetics, demographics, and existing guidelines for broodstock acquisition and mating designs can inform genetically cognizant hatchery management and stocking for striped bass in the Roanoke River drainage. In particular, we recommend the use of larger numbers of breeders and factorial mating designs to increase the genetic diversity of propagated striped bass stocked within the Roanoke River drainage.