The Neosho Smallmouth Bass Micropterus dolomieu velox is endemic to Arkansas River tributaries originating in the Ozark Highlands and Boston Mountains. Although morphologically and genetically distinct from other populations of Smallmouth Bass M. dolomieu, the conservation‐genetic status of Neosho Smallmouth Bass is largely unknown. To assist in filling this data gap, we quantified introgressive hybridization, genetic diversity, and population structure of Neosho Smallmouth Bass in two major river basins (Grand River and Illinois River) using seven polymorphic microsatellite markers. Introgressive hybridization with stocked Tennessee lake‐strain Smallmouth Bass was most prevalent in the Illinois River, wherein the overall genomic proportion of Neosho Smallmouth Bass alleles was only 0.422. After accounting for hybrid individuals, genetic diversity of Neosho Smallmouth Bass was generally higher in larger rivers and lower in smaller, isolated streams. Three distinct population clusters were identified at the uppermost level of genetic structure—one from the Illinois River basin and two from the Grand River basin. These three population boundaries accounted for approximately 7% of the hierarchical genetic variation within our data set, and substructure below the uppermost level accounted for an additional 2% of genetic variation. The population structure we discovered can provide a blueprint for management that conserves diversity within and among populations; for example, population boundaries can be used to determine brood source locations for supplemental stocking efforts to counter nonnative introgression. Introgressive hybridization with nonnative Smallmouth Bass appears to be the most pertinent threat to the Neosho Smallmouth Bass subspecies, which is of conservation value to the overall adaptability of Smallmouth Bass as a species.