The Ethiopian highlands – home to striking species diversity and endemism – are bisected by the Rift Valley, a zone of tectonic divergence. Using molecular data we examined the evolutionary history of two co-distributed species of African clawed frog (Xenopus clivii and X. largeni) that are endemic to this region. Our field collections substantially extend the known distribution of X. largeni, a species formerly known from highlands southeast of the Rift, but that also occurs to the northwest. In both species, analysis of mitochondrial DNA and 19 autosomal loci identifies significant population structure, suggests little or no recent migration across the Rift Valley, and provides divergence time estimates across the Rift of ∼1–3.5 million years. These results indicate that the Ethiopian Rift Valley is a major obstacle to dispersal of highland-adapted amphibians.