Xenopus gilli is a vulnerable anuran with a patchy distribution along the south-western coast of the Cape Province, South Africa. This species is sympatric with Xenopus laevis laevis, a widespread relative found over much of southern Africa. We examined the molecular phylogeography and population structure of the contact zone between these species to obtain information about historical biogeography and conservation management of this region. Analyses of the distribution, frequency, and cladistic and phenetic relationships among mitochondrial DNA haplotypes indicate that population subdivision is present in both taxa but that long-term isolation of sets of populations has occurred in X. gilli only. Haplotype and nucleotide diversity are also considerably higher within and among X. gilli ponds than X. l. laevis ponds in this region. We attribute the genetic segregation of X. gilli populations to ancient habitat fragmentation by ocean transgression into X. gilli habitat and to continued habitat alteration by human activity. The lower level of genetic diversity in X. L. laevis in this region is likely a result of a recent arrival of this taxon to the south-western Cape region relative to X. gilli. Population structure in X. l. laevis may be a result of isolation by distance. Clear evidence exists for at least two management units within X. gilli and strongly supports the establishment of protective measures east of False Bay in order to conserve a substantial portion of this species' extant genetic diversity.