Rose's mountain toad Capensibufo rosei survives in a few isolated montane populations in the south-western Cape of South Africa. It comprises several cryptic species but it is uncertain whether the lineage on the Cape Peninsula is distinct. We tested the hypothesis that toads from the Peninsula form a single genetic lineage but that different breeding sites are divergent at a population level as a result of limited dispersal abilities. Directed surveys were carried out to locate breeding sites and samples obtained were analysed in a phylogenetic and population genetic framework, using two mitochondrial markers. We found toads breeding at only one of five known historical breeding sites, although one new breeding site was also recorded. No toads were observed at 15 other non-breeding localities where they were historically observed. Toads from the two active breeding sites formed a single lineage that was sufficiently distinct to be given species status. However, these were discrete at a population level, with no shared haplotypes, suggesting no gene flow between sites. One site was particularly low in genetic diversity, implying increased vulnerability to stochastic events and elevated risk of extinction. These results, coupled with the failure to locate historically known sites on the Cape Peninsula, make this newly recognized Peninsula endemic a conservation priority. Efforts should focus on the protection and expansion of the two known surviving populations and the patches of habitat upon which they rely.