In this paper we support the division of the previously monotypic genus Ascaphus into two species based on an analysis of 23 allozyme loci from 34 populations. We use maximum likelihood to estimate the Ascaphus phylogeny from the allozyme data and find strongly supported monophyletic Rocky Mountain and Pacific clades. In a nonhierarchal, model-based cluster analysis of the data, each of the 1085 individual genotypes is correctly assigned to either Ascaphus montanus or A. truei with a high probability. We also find a virtually fixed difference between the species at the Pgm-2 locus. Within A. truei, we find a lack of significant pairwise F_ST values among populations from the Coast and central Cascades Mountains, suggesting relatively recent range expansion or contemporary gene flow among these populations. Olympic Mountains populations form a discrete clade in the allozyme topology and are fixed for a unique allele at the Lap locus. These populations remain isolated from the remainder of the species' range based on pairwise F_ST values. The four southernmost A. truei populations each show significant allelic divergence from the remaining populations (based on pairwise F_ST values), suggesting climate-induced isolation. In addition. we extend mtDNA sampling within the Rocky Mountains and sequence 530 nucleotides from the mtDNA Cytochrome b (cyt b) gene in 12 previously unsampled A. montanus populations. This additional sampling defines the geographic extent of a southern mtDNA clade distinguished on average by 0.024 substitutions per site from the northern clade. We use nested clade analysis, a coalescent-based divergence by isolation with migration model (MDIV), maximum-likelihood estimation of the mtDNA topology, and Bayesian model-based genotype assignment, to test predictions from two hypotheses: the western refugia hypothesis, which claims that A. montanus persisted in refugia west of the Snake River during Pleistocene glacial maxima, and the dual refugia hypothesis, which asserts that A. montanus occupied refugia within the Salmon and Clearwater River Valleys during glacial maxima. Our data do not support predictions of the western refugia hypothesis. Nested-clade analysis, estimated dates of lineage divergence supplied by MDIV, and the mtDNA topology support predictions of the dual refugia hypothesis; however, the allozyme topology fails to support some of these predictions. Allozyme and mtDNA data endorse the preliminary recognition of a minimum of two Evolutionarily Significant Units (ESUs) within A. truei: (1) populations from the Olympic Mountains and (2) populations south of the Umpqua River. Two ESUs are also suggested within A. montanus: (1) populations south of the South Fork of the Salmon River, and (2) populations to the north and west of the Salmon River (including the Blue, Wallowa and Seven Devils Mountains). The MDIV analysis indicates an exchange rate of 10 migrants per generation between the northern ESU and the southern ESU.