The increasing importance of the walleye (Sander vitreus) fishery in the New River, Virginia, and recent research findings motivated characterization of its genetic composition. Movements of radio-tagged fish suggested that walleyes living in Claytor Lake and the upper New River tend to spawn in different areas. In this study, allozyme, microsatellite DNA, and mitochondrial DNA genetic marker data were analyzed to assess population genetic differentiation among collections of New River walleye. The walleyes within Claytor Lake are a panmictic population, presumably resulting from years of stocking different genetic backgrounds and subsequent interbreeding. However, the genetic structure of walleyes from the New River shows the presence of more than one population. Fish in the New River system carry three previously unknown mitochondrial DNA haplotypes (43, 44, and 45), as well as high frequencies of characteristic alleles at particular microsatellite DNA loci. These observations may indicate a unique walleye stock that is native to the New River and which has remained spatially or temporally segregated by its spawning habits. The co-existence of different populations in the Claytor Lake / upper New River system justifies different management strategies. Management of the upper New River population should focus on conservation of the unique stock through marker-assisted selection of spawners with supplemental stocking of their offspring and/or strict harvest regulations.