The Ozark madtom, Noturus albater, is distributed in upland streams in the upper White, Black, and St. Francis rivers of Missouri and Arkansas, USA. Fixed chromosomal and biochemical differences between populations in the upper White River and the Black and St. Francis rivers led earlier researchers to suggest that the Ozark madtom comprised two distinct species. The current study reviewed previous data, and examined osteology, pigmentation, morphometrics and DNA sequences from the mitochondrial cytochrome b gene and the nuclear Recombination Activating Gene 2. Sequence data were analysed using parsimony and Bayesian methods to construct a phylogenetic hypothesis of relationships within Noturus and examine relationships of populations of Ozark madtoms. Results of the molecular analyses were consistent with earlier chromosomal and biochemical studies. Morphology could not distinguish between populations from the upper White River and populations from the Black and St. Francis rivers. However, karyotypes, allozyme variation, and DNA sequences all provide diagnostic characters demonstrating the presence of two species consistent with the phylogenetic species concept. We describe the populations from the Black and St. Francis rivers as Noturus maydeni, a new cryptic species of madtom catfish.