Barley ( Hordeum vulgare L.) is one of the most important cereal crops worldwide, with increasing demand for its production. However, during the long-term domestication of the cultivated barley, especially after the modern breeding and intensive cultivation, the genetic variation reduced significantly, leading to genetic erosion in this crop as well. A shorter growing season, more tolerant to soil salinity and drought than wheat are the advantages of this crop. Wild relatives of cultivated barley are potential source of valuable genetic materials for barley improvement. Genetic variation of wild species belonging to primary gene pool of barley is important in employing in barley breeding program, particularly for tolerance to biotic and abiotic stresses. Being a close relative of wild species ( H. spontaneum L.) and the lack of tolerance genes among the cultivated genotypes of barley makes gene introgression the appropriate avenue to transfer desired genes such as abiotic stress tolerance. In this research a backcross populations (BC 2 F 1 ) developed from interspecific hybridization between cultivated barley and its wild relative ( H. spontaneum L.) was assessed for genetic diversity using molecular markers which are valuable to assist background selection for recurrent parent. Results of cluster analysis (9 clusters) showed that only small number of the families from the population used (142 families) distantly clustered from their recurrent (cultivated) parent. Out of the 42 microsatellite (SSR) primers used, 23 were polymorphic and produced 123 bands. Polymorphism information content (PIC) ranged from 0.28-0.49 with an average value estimated for all loci 0.46. The highest PIC estimated for S2 and Xcinau167 markers shows that these markers are more efficient to differentiate backcross families. Principal coordinates analysis (PCoA) supported the results of clustering analysis. Arliquin and POPGENE softwares were used to analyze genetic diversity and molecular variance (AMOVA) among populations, respectively. Gst and Nm parameters had an average of 0.59 and 0.34, respectively, indicating a low gene flow among nine groups of backcross families. The AMOVA results showed that majority of genetic diversity belonged to within population variation (86.48%). Nei's genetic similarity ranged from 0.41 to 0.92 which were consistent with those of genetic distance. According to the obtained results, SSR and EST-SSR markers could be used as a valuable tool for acquiring molecular information and investigating genetic diversity. Furthermore, SSR markers have the potential to increase efficiencies of barley alien gene introgression programs in order to promote barley improvement particularly for tolerance to biotic and abiotic stresses. According to the results, since (1) the majority of genetic variation belonged to within the groups from the cluster analysis, (2) most of the families were grouped along with their recurrent parent, it can be concluded that microsatellite markers have strong differentiation ability to discriminate the resultant genotypes from a bi-parental cross. Keywords : Molecular marker, Genetic variation, Backcross population, Polymorphism, Genetic distance.