Walleye from the upper Cumberland River drainage were examined by means of mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis, microsatellite analysis of nuclear DNA, and sequencing of the mitochondrial DNA control region. The objectives of this study were to identify native Eastern Highland and introduced Great Lakes walleye and determine if introgression occurs between the two stocks. Additional collections were made from rivers of the Eastern Highlands to establish evolutionary relationships. Native and introduced walleye appear reproductively partitioned in the upper Cumberland River drainage, with natives persisting in the tributary Rockcastle River and introduced Great Lakes walleye reproducing in the Big South Fork of the Cumberland River. Additional partitioning between native and introduced stocks may occur in the Kanawha River, in the Ohio River drainage. Native walleye from the Cumberland River share a common ancestor with others from Eastern Highland rivers but are distantly related to populations from the southern Mobile River basin and the northern Great Lakes.