Recent studies have shown that the House Mouse (Mus musculus) has four subspecies in Iran. Although, these four subspecies have been recognized, the house mouse of east Iran showed high heterozygosity in various markers like allozymes, nuclear gene and mitochondrial gene sequences. Also, the taxonomy and diagnostic characters of mice populations in Iran and adjactment regions are poorly understood. In order to define clear characters for the subspecies described and identify the borders of Iranian subspeciese, thirty-one (31) populations were studied using three methods: chromosomal morphology, morphology and molecular analysis of the mitochondrial cytochrome b gene. Molecular analysis of the M. musculus samples revealed four clades: 1- clade M. m. isatissus (of Iran) and M. m. castaneus from (of India), 2- clade M. m. bacteriaus from eastern areas with higth intrasubspecies genetic distance, 3- clade M. m. domesticus in the Southern and western regions and 4- M. m. musculus in the northeastern region of Iran. Morphometric characters resulted in three groups that overlapped with each other. The morphological characters could not be separated M. m. isatissus and M. m. bacteriaus, from each other . Analysis of cytogenetic variables showed four clear groups better the molecular clads. In these methods, the central and eastern clades are two distinct groups that are well supported with difference in size of centromeric heterochromatin and their patterns. These results showed that cytogenetic studies are useful and easy methods for identify the diagnostic characters of Iranian subspeciese.