Genetic variation was assessed with protein electrophoresis for seven populations of the leopard darter, Percina pantherina, a species restricted to five rivers in the Little River System of southwestern Arkansas and southeastern Oklahoma. Analysis of genetic diversity indicated that only 10% of the total gene diversity (H_T = 0.03) reflects differences among populations; 7.3% was attributable to differences among populations in different rivers and 2.7% to differences among samples within rivers. Average heterozygosity was fairly low (0.020–0.031), but there was a relatively high level of allele diversity with two or more alleles detected at 21 of the 31 gene loci examined. Most polymorphism was due to alleles occurring at low frequencies (≤0.05) in restricted geographic areas. An allele frequency, maximum parsimony analysis revealed three primary clades: (1) populations in Little and Glover rivers, (2) populations from the Mountain Fork River drainage and (3) populations in Robinson Fork and Cossatot rivers. This pattern of geographic variation should be considered in future management of the species.