The Missouri saddled darter, Etheostoma tetrazonum, is endemic to the state of Missouri where it inhabits medium to large creeks and streams in the Ozark region. Two morphologically distinct forms of the species have been identified on the basis of color pattern in breeding males, one form restricted to the Meramec River drainage and another found in the Gasconade, Osage, and Moreau River drainages. Given the rather striking differences in color pattern, a molecular character set was assembled to determine the extent of genetic variation between these two forms and whether additional drainage specific patterns of divergence were present. Complete cytochrome b (cyt b) sequences were obtained from 36 individuals of E. tetrazonum representing 13 populations from throughout its range. Phylogenetic analysis of these data resulted in two, well-supported monophyletic groups within E. tetrazonum: one consisting of individuals from the Meramec River drainage and the other consisting of individuals from the Moreau, Osage, and Gasconade River drainages. These findings are consistent with the recognition of two species: one species from the Moreau, Osage, and Gasconade River drainages, and one restricted to the Meramec River drainage.