Walleye (Sander vitreus) is a freshwater species inhabiting a wide range of habitats across North America. Owing to its economic importance and popularity as a sportfish, this species has been widely introduced beyond its indigenous range. Previous genetic studies have revealed a genetically distinct strain of walleye in Mobile River Basin drainages, but little is known about its genetic structure and how this southern strain relates to northern walleye populations. Another unresolved question is the hybridization status of walleye in Mobile River Basin following northern walleye introductions. In this study, we used genotyping-by-sequencing (GBS) data from 60 walleye individuals to infer genetic diversity and structure among northern and southern (Mobile River Basin) walleye populations. With this GBS data, a SNP assay with 68 diagnostic markers was developed for rapid and accurate identification of genetic purity and classification of various (northern/southern) hybrid classes among walleye individuals.