Eastern broad-toothed field mouse, Apodemus mystacinus, is a rocky habitat dwelling rodent distributed in Asia Minor, the Levant, the Caucasus, and the Zagros Mountains. In this study, we investigated the phylogenetic relationship between different populations of A. mystacinus throughout its range, based on the mitochondrial cytb marker. Phylogenetic analyses revealed the existence of five separately evolving lineages within A. mystacinus, of which two previously unrecognized lineages were identified in the Zagros Mountains and the Levant. Divergence between two major clades of the subgenus Karstomys, corresponding to A. mystacinus and Apodemus epimelas, is inferred to coincide with the Messinian Salinity Crisis (Late Miocene), whereas the splits between major lineages of A. mystacinus are inferred to have occurred during the Pleistocene. Colonization of the Zagros may have occurred from different refugia via eastward migration of the Turkish population and then again by a more recent colonization from the Caucasus, after reopening of the land corridor between the Caucasus and the Zagros Mountains during the Holocene drought.