Barking frogs (Eleutherodactylus augusti) are distributed from southern Mexico along the Sierra Madre Occidental into Arizona and the Sierra Madre Oriental into Texas and New Mexico. Barking frogs in Arizona and most of Texas live in rocky areas in oak woodland, while those in New Mexico and far western Texas live in rodent burrows in desertscrub. Barking frogs in each of the three states have distinct coloration and differ in sexually dimorphic characters, female vocalization, and skin toxicity. We analyzed advertisement call variation and conducted a phylogenetic analysis using mitochondrial DNA sequences (ND2 and tRNA regions) for barking frogs from these three states. Advertisement calls of frogs from Arizona were significantly longer in duration, higher in frequency, and had longer duration pulses than those of frogs from either New Mexico or Texas; frogs from these latter two sites were indistinguishable in these call variables. Phylogenetic analysis showed deep divisions among barking frogs from the three states. Differences in call structure, coloration, and mitochondrial DNA sequences strongly suggest that barking frogs in Arizona are reproductively isolated from those in New Mexico and Texas. Our results indicate that either northern populations are connected via gene flow through southern Mexico (i.e., they are subspecies as currently recognized), or represent independent lineages as originally described (i.e., western barking frogs, E. cactorum in AZ, and the eastern barking frogs, E. latrans in NM, TX).