Sequences of a mtDNA gene, cytochrome b, were used to generate a phylogenetic hypothesis for the evolution of Iberian archaeobatrachian frogs of the genus Discoglossus. Results from the inferred phylogenetic branching pattern and the sequence divergence among species and populations support the following: (1) the two endemic species from Iberia, D. galganoi and D. jeanneae are sister taxa and are reciprocally monophyletic with respect to their mtDNA; (2) the geographic distribution of these two morphologically cryptic species differs considerably from previous expectations. D. galganoi is limited to the metamorphic granitic and shale substrates of western Spain and Portugal, while D. jeanneae extends over the limestone and gypsum substrates of most of eastern Spain from the Strait of Gibraltar to the Pyrenees; (3) sequence divergence between D. galganoi and D. jeanneae averages 8.6%, suggesting that the two lineages diverged before the Mio–Pliocene boundary. We suggest that these lineages were isolated by the opening of the Betic Strait and that isolation has been maintained until recently by the Guadalquivir River Basin; (4) all specimens of D. jeanneae, from the central Pyrenees in the north to the southern end of the Iberian Peninsula have nearly identical cytochrome b haplotypes, clear evidence of a rapid relatively recent (Late Pleistocene–Quaternary) expansion of the species; (5) populations of D. pictus from northeastern Spain are highly divergent from all other Discoglossus studied and do not fall within the Iberian clade.