Two species of brush-tailed mice, genus Calomyscus, are known from eastern Iran: Calomyscus hotsoni has been reported from southwestern Pakistan and southeastern Iran, and C. elburzensis ranges through northeastern and central Iran. Based on molecular studies of two mitochondrial genes, all the specimens from eastern Iran examined herein belong to either of these two species. Furthermore, our data expand the northern distribution limits of C. hotsoni to just south of Birjand and the southern limits of C. elburzensis to east of Birjand. Morphometric analyses conducted on three geographic groups of C. hotsoni within Iran, contained specimens from Birjand (group 1), Zahedan and Khash (group 2) and Saravan (as group 3) revealed a north-to-south cline of decreasing body and cranial size, such that the most significant differences were between the northern and southern most groups. Karyological studies also showed differences in autosomal arms between the two geographical groups in Iran. Although the phylogenetic analyses separated these two groups into distinct clades, along with a third clade containing most of the C. hotsoni from Pakistan. The morphometric and molecular partitioning of geographic populations of C. hotsoni were not concordant. We consider the north and south groups of C. hotsoni as distinct Evolutionary Significant Units. There is evidence of introgression between the two forms across a broad geographic area presented by individuals of group 2 resulting in a clinal pattern of variation.