Wheat is the major source of energy, protein and fibers in human nutrition and is the most important crop in the world. Wild relatives of cultivated wheats are potential sources of valuable genetic materials for wheat improvement. Due to the considerable area of the cultivated wheat in Iran, it is useful to determine the genetic relationships among cultivars and genotypes of this plant, in order to preserve its genetic variation, recognition and the selection of the desired genotypes to improve it. Genetic variation of wild species belonging to primary gene pool of wheat is important in employing in wheat breeding program particularly for tolerance to biotic and abiotic stresses. The goal of breeding programs is introducing of new varieties with high yield and compatibility. To receive this goal, it is necessary to identify genetic diversity in interspecies and interspecies. Among the previous molecular markers used in wheat, microsatellite markers showed higher efficiency for separating the wheat genotypes, due to the codominant inheritance nature, high reproducibility and having high levels of polymorphism. therefore, In the present study, genetic variation of inter and intra species of Triticum monococcum . aegilopoides , Aegilops tauschii and Ae cylindrica was investigated using 20 polymorphic microsatellite (SSR) markers. Data from scored bands were analyzed by Power marker, Arlequin and NTSYS softwares.Twenty polymorphism microsatellite (SSR) markers in 21 studied genotypes amplified 180 alleles in total by an average of 9 alleles per locus. Genetic variation for all loci ranged from 0.74-0.90 with an average value of 0.83. Polymorphism information content (PIC) ranged from 0.7-0.89 with an average value computed for all loci of 0.82. The highest PIC estimated for Xgwm186 and Xgwm205 markers shows that these markers are more useful in genotypic differentiation. Major allele frequency ranged from 0.14-0.39 with an average of 0.27. Dendrogram resulting from the neighbor-joining metod in which the tree divided to the genotypic groups, correspond to Ae. tauschii , T. monococcum . aegilopoides and Ae. cylindrica . The results indicated that T. monococcum . aegilopoides and Ae. tauschii were more closely related, while Ae. cylindrica was more distantly related to other two species. Analysis of molecular variance revealed a significantly greater within species genetic variation than between species, where 76.4% of the computed genetic variation belonged to within species and the remaining belonged to between species )23.6% ). In calculation of cophenetic correlation coefficient by NTSYS software, the highest cophenetic coefficient related to Jaccard's similarity coefficient and UPGMA method by r=0.90. In adjusted principle coordinate analysis (PCoA) the three first components explaid 25.71 of the total variation, which implies that the used markers possessed a suitable dispersion of markers in the genome. It is hence concluded that SSR markers are not only useful in differentiating wild species of wheat (A, B, C and D genomes) but also in assessing genetic variation of genotypes within species. Futhermore, SSR markers have the potentials to increase efficiencies of various wheat alien gene introgression programs in order to promote wheat improvement particularly for tolerance to biotc and abiotic stresses. Keyword : genetic variation, wheat