A major challenge faced by conservation officials is determining which habitats are most vulnerable to anthropogenic perturbation and thus are in most critical need of protection. We utilized a fish with life history attributes presumably conducive to low rates of gene flow and small effective population size to gain insight into the appropriate conservation units in the Carolina Sandhills, a threatened ecosystem in the southeastern United States. We assessed variation in nuclear and mitochondrial DNA sequence data throughout the range of the Pinewoods Darter, Etheostoma mariae (Percidae), a species endemic to headwater streams of the Lumber and Little Pee Dee rivers (Pee Dee Drainage) in the Carolina Sandhills. Concordant partitions in nuclear and mitochondrial DNA loci support the designation of two Evolutionarily Significant Units (ESUs): one each from Lumber and Little Pee Dee rivers. The data suggest these populations have been historically isolated and are on distinct evolutionary trajectories. Additionally, this research underscores the potential importance of fine-scale sampling in conservation genetics studies of organisms predisposed to genetic differentiation and demonstrates that significant population structure can occur even within a single drainage.