Remnant populations of leopard frogs within the Virgin River drainage and adjacent portions of the Colorado River (Black Canyon) in northwestern Arizona and southern Nevada either represent the reportedly extinct taxon Rana onca or northern, disjunct Rana yavapaiensis. To determine the evolutionary distinctiveness of these leopard frogs, we evaluated mitochondrial DNA (mtDNA) restriction site variation (RFLP), mtDNA control region sequences, randomly amplified polymorphic DNA (RAPD) markers, and morphological characters. Individuals from the Virgin River drainage and Black Canyon represented a single RFLP haplotype and were identical for nucleotides along a portion of control region sequence. Evaluations of RAPD data demonstrated high levels of similarity among individuals and populations from this region. Leopard frogs from the Virgin River drainage and Black Canyon differed from R. yavapaiensis from west-central Arizona and northern Mexico in maximum parsimony and distance analyses of RFLP and control region sequence data and in maximum-likelihood analysis of the sequence data. Multidimensional scaling of RAPD data provided a similar and congruent indication of this separation. Analysis of principal component scores demonstrated significant morphological differentiation between leopard frog specimens from the Virgin River drainage and R. yavapaiensis. Parallel patterns of divergence observed in the mtDNA, RAPD, and morphological analyses indicate that leopard frogs from the Virgin River drainage and adjacent portions of the Colorado River are phylogenetically distinct. These leopard frogs should be recognized as a lineage separate from southern populations of R. yavapaiensis and classified as the species R. onca.