The spiny mouse Acomys cilicicus is confined to a similar to 120 km(2) coastal strip between Silifke and Erdemli in southern Turkey. The species has long been of uncertain taxonomic status. We sequenced a 950-bp fragment of the mitochondrial cytochrome b (CYTB) gene from 16 individuals of A. cilicicus across five localities and integrated these data with published CYTB sequences of A. minous, A. nesiotes andA. cahirinus. Median-joining network and Kimura-2-parameter distance analyses revealed two geographically structured haplotypes of A. cilicicus within an overall low diversity background (< 2% divergence). Phylogenetic reconstructions, using maximum likelihood and Bayesian relaxed-clock approaches, identified A. cilicicus as a strongly supported, monophyletic sister clade to A. minous. Divergence dating placed the split between A. cilicicus and A. minous at similar to 0.17 million years ago (95% highest posterior density: 0.03-0.37 Mya). Coalescent-based migration modelling (MIGRATE) supports a two-stage colonisation scenario: an ancestral dispersal ofA. cahirinus into Crete giving rise to A. minous, followed by Pleistocene maritime colonisation from Crete to southern Anatolia, yielding A. cilicicus. These results validate the distinct species status of A. cilicicus as a discrete conservation unit and highlight the need for multilocus nuclear markers to resolve incomplete lineage sorting in this recently diverged Mediterranean clade.