The Barrens Darter, Etheostoma forbesi, is one of the most geographically restricted freshwater fish species in North America, with a distribution limited to headwater portions of nine streams in the western part of the upper Caney Fork, a tributary of the Cumberland River in Tennessee. This limited geographic distribution makes Etheostoma forbesi especially vulnerable to potential threats posed by human alterations to rivers and streams, and the risk of ecological competition and introgressive hybridization with the closely related Fringed Darter, Etheostoma crossopterum. Museum collection records and targeted surveys conducted since its description suggest that present-day range of Etheostoma forbesi does not include several streams it previously inhabited—some as recently as 15 years ago. We investigate the geographic distribution and variation in meristic traits of both Etheostoma forbesi and Etheostoma crossopterum in the upper Caney Fork system through the examination of all available museum vouchers, and assess phylogeographic patterns among populations using mitochondrial DNA sequence data. We report a newly discovered population of Etheostoma forbesi from the upper Collins River, and present evidence that Etheostoma crossopterum has displaced Etheostoma forbesi in the Hickory Creek system and Mud Creek in the upper Barren Fork River system. There are no differences in scale counts between the two species, but the sum of the number of rays in the anal, pectoral, and second dorsal fins is diagnostic. Analysis of mtDNA variation indicates gene flow between Etheostoma forbesi and Etheostoma crossopterum. Our results suggest that sympatry of the two species in the western upper Caney Fork system is a potential threat to the persistence of Etheostoma forbesi.