In the present study, 37 microsatellite primerpairs were used for germplasm analysis and estimation of the genetic relationship and diversity between 21 spring and winter genotypes of Persian breed wheat. Genetic diversity (DI) and polymorphism information content (PIC) were calculated with average 0.68 and 0.63, respectively. Genetic similarity was calculated with Nei & Li and Jaccard methods with average 0.201 and 0.325, respetcively. The highest levels of genetic similarity for genotypes were found in Ghods and Alvand genotypes by two methods and the lowest was in Bolani and Niknejhad with Nei & Li and Jaccard methods.The number of alleles per microsatellite marker varied from 2 to 10 (average 5.37) and a total of 199 alleles were detected. The result showed all of the genotpyes could be distinguished and clustered into 4 groups with Nei & li similarity and UPGMA clustering method and all cultivars segregated by growgth type, i.e spring and winter. The data suggest that microsatellite can be used to estimate high and repeatable Polymorphism genetic diversity.