Conservation and management of cryptic species is particularly challenging since it can be difficult to determine their exact distributions. Knowledge of species distribution is required to recognize management units based on taxonomy and whether there are any pertinent biogeographic patterns that could be relevant to the development of a management plan. Such a situation exists in Illinois with the Rosyface Shiner Notropis rubellus and Carmine Shiner Notropis percobromus. There are no reliable anatomical characters to distinguish between the two species. Instead, they are distinguished by genetic data. Samples were obtained for these two species from across the state from each major watershed in which they are found. Based on cytochrome b sequence variation the two species can be distinguished by 44 species-distinct differences. Notropis percobromus is restricted to the Rock River watershed and nearby Mississippi River tributaries in northwest Illinois. Notropis rubellus is found in the Illinois River basin and Vermilion-Wabash watershed. This is a reflection of historical drainage patterns from the geologic period when eastern Illinois was connected to the Great Lakes, as opposed to today when all the rivers in this study are part of the Mississippi basin. Using a previously published rate of divergence of the cytochrome b gene, we estimated a divergence time for these two species that was consistent with a previous estimate—2.8–2.6 MYA. Statistically, there was no genetic difference among populations within N. rubellus or N. percobromus. Haplotype networks and a phylogenetic analysis do provide some evidence for potential bottlenecks/founder effects and/or haplotype-specific selection within each species.