Patterns of phylogeography and gene flow were examined in the canyon treefrog, Hyla arenicolor. A total of 973 bp of mitochondrial cytochrome b sequence data were obtained for 65 individuals from 53 populations, yielding 50 unique haplotypes. Interpopulation sequence variation ranged from 0 to 13.7%. Phylogenetic analysis revealed three deeply divergent mtDNA lineages. These three Clades were mapped onto geography and found to represent completely concordant, nonoverlapping, geographical regions. Levels of sequence divergence between the three Clades were equal to or greater than published levels of divergence found in other vertebrate species and genera. Furthermore, one Clade of H. arenicolor was found to be more closely related to the outgroup H. eximia than to other H. arenicolor, suggesting that the taxonomy of this species may require revision.