Brush-tailed mice, family Calomyscidae, are endemic to southwestern Asia and are represented by eight described species and three additional lineages. Although this family includes only a single genus, Calomyscus, in which several molecular phylogenies were recovered as a monophyletic clade, no updated evolutionary survey has been undertaken until now. We present a time-calibrated molecular phylogenetic tree of members of this genus using mitochondrial and nuclear markers. According to our results, divergence of calomyscid species occurred between 5.85 and 3.35 Mya during the Late Miocene and Pliocene. It seems that the species that occur in the eastern part of the range of the family diverged earlier (highest posterior density (HPD) 3.95–7.28 Mya) from congeners than those from the western part (HPD 2.18–4.24 Mya). Furthermore, we provide new insights on a poorly known species, C. grandis, endemic to the Elburz Mountains, including its phylogeny, morphology (geometric morphometric, karyology, and traditional morphometric methods), and distribution. Our findings show greater haplotype diversity than previously reported and extend the known range of C. grandis in northern Iran. Molecular data detects the existence of an additional distinct lineage from the Zagros Mountains, which appears to be differentiated from other lineages of Calomyscus at levels observed between species. Morphologically, compared to other species, the new lineage is the largest brush-tailed mouse and shows a number of remarkable differences in cranial features (e.g., widest cranium and highest mandible) with chromosomal component 2n  = 44 and FNa = 62. Higher species diversity of calomyscids around the Iranian Plateau may be the result of geological events and the subsequent aridification during the Pliocene. The Lut Desert and the Zagros Mountains provided barriers and refugal habitat, respectively, that likely contributed to speciation events within the genus. Additional sampling may discover more distinct lineages in the Zagros Mountains or Anatolia and the eastern Mediterranean region.

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