Walleye (Sander vitreus) is a popular sportfish threatened by overexploitation, habitat destruction, and loss of genetic integrity due to non-native walleye stocking. Previous studies have identified a genetically distinct lineage of walleye in the Mobile River Basin, but further work is needed to assess population structure and introgression among this southern lineage and northern populations. Here we generated 2782 genome-wide single nucleotide polymorphisms (SNPs) to characterize the genetic uniqueness of southern walleye. We also found strong evidence for a historical declining population trend with reduced genetic diversity and effective population size in a southern walleye population of conservation importance. A 68-SNP panel was developed for rapid identification of genetic integrity and hybrid classification among northern and southern walleye, enabling us to identify an anthropogenic hybrid zone resulting from the previous introduction of northern walleye into the Black Warrior River drainage, Alabama. Our results highlight the need for conservation management of southern walleye in the Mobile River Basin, with our 68-SNP assay already being implemented in ongoing stream survey and captive breeding programs.