The Smallmouth Bass (Micropterus dolomieu; SMB) is a widely distributed black bass species, but the southwestern edge of the species range within the Interior Highlands contains some of the most divergent ecotypes. The Neosho subspecies (M. d. velox) inhabits tributaries of the Arkansas River within the Ozark Mountains and a second lineage is reported from drainages of the Ouachita Mountains. We sought to develop a single nucleotide polymorphism (SNP) panel to (1) diagnose hybridization with sympatric Spotted Bass (Micropterus punctulatus; SPB) and non-native Northern SMB (M. d. dolomieu) stocked in the region, and (2) delineate population structure within the ranges of the Neosho and Ouachita SMB lineages. We obtained 76 individual SMB samples from across their range but concentrated within the Interior Highlands (n = 50). We also included 3 SPB to allow for hybrid detection and 3 Shoal Bass (Micropterus cataractae) as an outgroup. Phylogenetic trees constructed with the generated SNP data corroborated the existence of at least three major lineages of SMB (Northern, Neosho, and Ouachita), each containing varying degrees of differentiation among major drainages. Simulation analyses revealed that chosen SNPs had high power (> 0.9) to assign SMB × SPB hybrid categories and similarly high power (> 0.8) for Northern SMB × Interior Highlands SMB hybrids. Clustering methods delineated major inter-basin population structure within the native ranges of Neosho and Ouachita SMB with chosen SNPs. Anticipated uses of the resulting 192-loci SNP panel include conservation planning, fisheries management assessments, and ecological investigations of the Neosho and Ouachita SMB lineages.