Objective: Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive neuromuscular disorders caused by defect in the human dystrophin gene. DMD is the most severe and frequent inherited disease affecting about 1:3500 male births. Disease is progressive and lead to death in third decades of life. Since DMD/BMD are incurable and there is no effective treatment, therefore, prevention of disease is important and strongly depends on determination of carrier status in the at risk women. Currently several complicated methods are used for carrier detection which are time consuming, expensive and need special equipment. Moreover, There are numbers of polymorphic DNA markers within the dystrophin gene which can be used in linkage analysis of DMD/BMD for carrier detection and prenatal diagnosis. In comparison with other expensive and sophisticated methods such as DHPLC, MLPA, FISH and Q real-time PCR, linkage analysis is easy, rapid and cost benefit and also there is no need to indentify the mutations. While, allele frequency and hetetrosygosity of these polymorphic DNA markers have been determined in different populations and ethnic groups. But, there is no reliable published data in Iranian populations . In this study we investigated heterozygosity and allele frequency of three polymorphic restriction sites and also four highly polymorphic (CA) n repeat microsatellite loci (STRs) within hot spots region of human dystrophin gene in 60 healthy Iranian. Materials and Methods: Using the polymerase chain reaction (PCR) we determine allele frequencies and heterosygosity of seven polymorphic loci including pERT87-8/TaqI, pERT87-15/BamH1, pERT87-15/XmnI, STR44, STR45, STR49 and STR50 in 60 healthy Iranian populations. Results: The results of PCR-RFLPs analysis showed that the pERT87-15/XmnI locus had the most hetrosygosity (47.17%) among of three restriction site polymorphisms and pERT87-8/TaqI had the least heterosygosity (35.84%). Also our data on the three restriction systems in the Iranian showed the most allelic frequency for F2 allele (0.779) in pERT87-15/BamHI locus. Moreover, our results on four polymorphic (CA) n repeat indicated that STR49 had most alleles number (8) and also had the most heterosygosity (35.9). STR44 had the least alleles number and also least heterosigosity. Conclusion: Compare to the other populations there was difference in the distribution of allelic polymorphism frequencies and heterosygosity in Iranian population. We identified pERT87-8/TaqI, pERT87-15/BamHI, pERT87-15/XmnI loci along with STR49 had high heterozigosity and their combination are useful in linkage analysis of Iranian DMD families in both carrier detection and prenatal diagnosis.