The Nocomis biguttatus species group ranges widely across North America from the Red River in Oklahoma and Arkansas north to Minnesota and east–west from Wyoming to Ontario. The group includes three traditionally recognized allopatric species: the wide-ranging N. biguttatus and two geographically more restricted species, N. asper from the western Ozarks (Arkansas River system) and two disjunct locations in the Red River system, and N. effusus from the Green, Cumberland, and lower Tennessee rivers. Separate analyses of the mitochondrial cytb gene and two nuclear genes (S7 intron 1 and a portion of the gene for growth hormone, GH), each resolved a cryptic species previously treated as N. biguttatus from the southern Ozarks (White River). Relationships among the four species were unresolved because of conflicts between cytb and S7 and a lack of resolution for GH. A previously indicated N. biguttatus–N. effusus sister-relationship appears to reflect past hybridization and mtDNA capture by N. effusus. Nocomis biguttatus includes four primary cytb clades with unresolved inter-relationships. A Northern Ozarks–Great Plains–Upper Midwest Clade and an Ohio River–Eastern Great Lakes Clade presumably represent late Quaternary dispersal from glacial refugia in, respectively, the northern Ozarks and an unglaciated portion of the Ohio River system. Other clades include one from the Meramec River and a Black River– St. Francis River Clade. There was evidence in N. effusus for a phylogeographic break between the lower Tennessee River and the Green-Cumberland basins. Geographic structure is weak in N. asper, indicating relatively recent contact between now disjunct populations in the Arkansas and Red river basins. The Blue River population of N. asper appears to reflect late Pleistocene or Holocene hybridization and genetic swamping of a resident native population of N. biguttatus by an invading population of N. asper. This postulates past occurrence of N. biguttatus far south of its present range.