Cytotaxonomy is a branch of cytogenetics, devoted to the comparative study of karyological features for systematic and evolutionary purposes. Surely, awareness of chromosomal characters increases our knowledge for a variety of different fields of study. In this study, Cytogenetic analyses were performed in 92 Mus musculus specimens from 26 localities in Iran. Cytogenetic characteristics of the house mouse, Mus musculus, in Iran show that the chromosome number is 2n=40 and the arm number is NF=40. The karyotype consisted of 40 Acrocentric (A) chromosome pairs. The L/S (r ratio) was between 2.0621 – 4.5862. The Length of shortest Chromosome, Length of longest Chromosome and Mean of chromosomal Length were between 2- 3.58 ,6.07-7.01 and 3.43- 5.05 (μm) respectively. The results show two distinct katyotypic formulae were found, namely cytotype B and cytotype C . Asymmetry indexes (AI, DI, As%, A, A2, A1 and Syi%) in all population except Birjand and Khash showed symmetry in chromosomal. In clustering methods we are four cluster ,one is for specimens of central and east of Iran, second cluster is for specimens from south and west of Iran ,third cluster is for eight specimens of Birjand and finally fourth cluster is for two specimens of Kash locality

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