Darters represent one of the most diverse groups of freshwater fishes in North America, but approximately 33% of the 215 recognized species are considered imperiled on the IUCN Red List. Discovery and description of new darter species continues at a relatively rapid pace, with many exhibiting microendemic geographic distributions and little baseline data for conservation decisions. The Blueface Darter Etheostoma cyanoprosopum is a newly described species that occupies < 20 km of stream reaches in the Bear Creek system of the Tennessee River drainage and the Hubbard Creek system of the Black Warrior River drainage in the Mobile Basin (Alabama, USA). In addition to restricted distribution, the species is threatened by habitat degradation and several natural and man-made barriers that putatively fragment connectivity among populations. This study used microsatellite and mitochondrial (mt) DNA data with comparisons to the relatively broadly distributed sister species (Bandfin Darter E. zonistium) to evaluate multiple objectives about genetic connectivity and diversity among Blueface Darter populations. Analysis of mtDNA data indicated a lack of historical structuring across the Tennessee-Black Warrior river drainage divide and within the Bear Creek system. However, microsatellite-based Bayesian cluster analyses and F-statistics suggested contemporary isolation across the drainage divide and evidence for reservoir-induced fragmentation within the Bear Creek system. Compared to the sister Bandfin Darter, Blueface Darter populations exhibited reduced levels of genetic diversity, rendering them more susceptible to local extirpation and reduced fitness. Continued monitoring and quantitative ecological studies are recommended to understand population-specific measures of occurrence and abundance.