Iran is located in the southeastern part of Fertile Crescent where is considered as the center of origin and domestication of cultivated barley in the world. Therefore, having information about genetic diversity of this species is very important. In order to evaluate genetic variation in Hordeum vulgare L. cultivated in Iran, 14 accessions including seven of two-rowed (21 individual) and seven six-rowed barley  (21 individual) accessions from various regions of Iran and one accession of commercial cultivar ‘Morex’ were evaluated using 20 primer pairs of microsatellites. Microsatellite data were analyzed by Powermarker software ver. 3.25. The results of this study showed high genetic diversity (mean polymorphism information content or PIC = 0.670 and mean allele number = 6.65) in Iranian barley landraces. The genetic diversity in var. distichon was considerably higher than var. hexastichon.