Calomyscus elburzensis Goodwin, 1938, was previously known from central and northern Iran to southern and southwestern Turkmenistan and northwestern Afghanistan. Its type locality is at altitude of 1200m elevation in the Kurkhud Mts., North Khorasan province in northeastern Iran. This study provides new evidence that its range is more extensive than thought previously and central Iranian Mountains, Shirkuh (Yazd province)and Karkas Mts. in Isfahan and Zanjan provinces in the northwest were recognized as its distribution areas. Two major clades were detected within C. elburzensis from the analysis of two mitochondrial genes Cytb and CoI. The outline on m2 and karyotypes analyses also supported these two molecular clades; one northeast with 2n=44 and FNa=58, 60 and 62 and the other west and northwest of central Iranian desert with 2n=44 and Fna=70.The Statistical Dispersal-Vicariance Analysis (S-DIVA) suggested dispersal events from north east to the center of Iran occurred in two separate occasions. As 2.5% was the rather low level of sequence divergence between these two major clades, we assigned the central-northwestern brush-tailed mice as a subspecies of C. elburzensis and therefore we named as C. e. isatissus ssp. novo.