As a popular sportfish and key aquatic predator, Walleye populations must be carefully managed to support ecosystem health and a sustainable recreational fishing industry. Two genetically distinct strains of Walleye (Sander vitreus) inhabit Kentucky watersheds: the Great Lakes (GL) strain and the native Eastern Highlands (EH) strain. Previous stocking efforts have led to the GL strain entering EH strain waterways and causing hybridization. This study assesses the current status of wild Walleye genetics in Kentucky and evaluates the accuracy of genetic testing methods. The objectives were to identify wild Walleye strains using single-nucleotide polymorphisms (SNPs) and to evaluate two newly developed SNP assays (Figure 1). Fifty Walleye fin clips from three waterways were collected by the Kentucky Department of Fish and Wildlife (KDFW) in Spring 2025 (Figure 2). DNA was extracted using Promega Wizard® Genomic DNA Purification Kit, quantified with a NanoDrop 2000 spectrometer, standardized to 10 ng/µL, and analyzed using qPCR genotyping on a 7500 Real-Time PCR system using two SNP assays. The EH probe was VIC dyed, and the GL probe was FAM dyed in both assays. Thirty samples were identified as EH strain, 19 as GL strain, and one as a hybrid (Figure 3). Additional genetic analysis will be conducted to further validate the accuracy of SNP assays using fragment analysis based on 7 polymorphic microsatellite loci.