Candy darter Etheostoma osburni, a federally endangered non-game fish, has been extirpated from most of its historic range in Virginia and now occurs in four isolated populations in the New River drainage. Understanding of population genetic structure will provide insights into the recent natural history of the species and can inform conservation management. Our objectives were to: characterize population genetic structure, estimate and compare effective population sizes (N_e), and use this information to infer recent population history. Variation at mitochondrial cytochrome b sequences among 150 individuals showed 10 haplotypes separated by 1–14 mutational steps, some shared and some unique to particular populations. Variation at 12 microsatellite loci among 171 individuals showed lower variation in Dismal Creek than in other populations. All populations showed evidence of having experienced a genetic bottleneck and were highly differentiated from one another based on both types of DNA markers. Population genetic structure was related to stream position in regard to the New River, suggesting that populations were once connected. N_e estimates for all populations were less than the 500 recommended to maintain evolutionary potential, but most estimates were greater than the 100 needed for use as source populations. Our findings indicate that habitat management to allow expansion of populations, and translocations to exchange genetic material among populations, may be effective tactics to promote conservation of candy darter in Virginia.