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The tailed frog Ascaphus truei Stejneger, 1899 is the most primitive extant anuran and the sister taxon to the clade of all other living frogs. The species occupies two disjunct ranges in the Northwest region of North America: the Cascade Mountains and coastal area from British Columbia to Northern California, and an inland range in the northern Rocky Mountains and the Blue andWallowa mountains. A previous study led to the isolation of eight peptides with antimicrobial activity (termed the ascaphins) from skin secretions of A. truei from the coastal range. The present study has used peptidomic analysis to identify the products of orthologous ascaphin genes in electrically-stimulated skin secretions from inland range specimens. Structural characterization of the peptides demonstrated that ascaphins from the inland range contained the following amino acid substitutions compared with orthologs from the coastal range frogs: ascaphin-1 (Ala12→Glu), ascaphin-3 (Asp4→Glu), ascaphin-4 (Ala19→Ser), ascaphin-5 (Lys12→Thr), and ascaphin-7 (Gly8→Ser and Ser20→Asn). Orthologs of ascaphins-2, -6, and -8 were not identified but a paralog of ascaphin-5, identical to ascaphin-5 from coastal range frogs, was found. The data support the claims, derived from analysis of the nucleotide sequences of mitochondrial genes, that the inland populations of the tailed frog should be recognized as a distinct species, the Rocky Mountain tailed frog Ascaphus montanus and that the divergence of the species from A. truei probably occurred in the late Miocene (approximately 10 Mya).