The identification of subterranean species at risk and the development of management strategies face a host of challenges such as inaccessible habitat, a lack of basic ecological information, and, in the case of aquatic species termed stygobionts, groundwater contamination and withdrawal. In light of these challenges, genetic data may inform management and conservation decisions. We present here results from an analysis of population structure of the stygobiont, Antrolana lira, from nine geographic locations spanning two-thirds of the species' entire range in the Shenandoah Valley of Virginia and West Virginia. A 658 bp fragment of DNA coding for the cytochrome oxidase c subunit I (COI) gene was sequenced from 70 individuals, yielding 14 haplotypes. Phylogenetic analysis of the 14 haplotypes showed the presence of three distinct clades which corresponded to three geographic groups. Divergence levels among sequences within sites and among sites within clades was low (uncorrected “p”  =  0.00 to 0.42%), but were much higher among sequences between clades (uncorrected “p”  =  9.53 to 11.24%). AMOVA corroborated these findings, partitioning over 99% of the sequence variation to differences among clades. The genetic structure of A. lira clades seemed to largely mirror the geologic structure of the area, with potential subterranean hydrologic barriers separating the clades. Geographic populations within each clade likely constituted only one or two demes. We suggest that these clades remain at risk from anthropogenic disturbances such as urban development, and propose that each of the clades be considered a distinct management unit in order to preserve the total genetic diversity for the species.