I used meristic, mitochondrial DNA, and nuclear DNA methods to infer the most likely ancestry of a putative hybrid specimen of Percina captured in the Roanoke River of Virginia. Potential parental species included Percina rex, P. nevisense, and P. roanoka. All nine of the meristic characters that I counted for the putative hybrid were within the published ranges of counts for P. nevisense, whereas counts for five characters were outside of published ranges for either P. roanoka or P. rex. These results were consistent with pure P. nevisense ancestry as well as various hybridization scenarios. Based on analysis of 1037 bp of the ND2 mitochondrial gene, the haplotype of the putative hybrid was identical to a known P. rex haplotype, but <86% similar to the closest-matching haplotypes for either of the other two species. Bayesian admixture analysis using seven nuclear microsatellite markers indicated a high probability of P. rex or P. nevisense ancestry and a low probability of P. roanoka ancestry. Taking all evidence together, the most parsimonious explanation is that the specimen was a hybrid between P. rex and P. nevisense.