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, and mitochondrial gene sequences. Moreover, the taxonomy and diagnostic characters of mice populations in Iran and adjacent regions are poorly understood. To define evident characteristics for the subspecies described and identify the borders of Iranian subspecies, thirty-one 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 (of India), 2- clade M. m. bactrianus from eastern areas with high 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 separate M. m. isatissus, and M. m. bactrianus, from each other. Analysis of cytogenetic variables showed four clear groups better than molecular clades. In these methods, the central and eastern clades are two distinct groups that are well supported by the difference in the size of centromeric heterochromatin and their patterns. These results showed that cytogenetic studies are useful and easy methods for identifying the diagnostic characters of Iranian subspecies.

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