Abstract: The genetic diversity among cultivated barley with different responses to drought stress was investigated. Initial screens involved growing 16 cultivars under drought conditions in greenhouse. These tests involved 5 tolerant and 5 sensitive types. The results of RAPD analysis indicated its suitability for determination of polymorphism among the samples. Among 30 primers used with RAPD-PCR technique, primers No. 3, 26 and 28 did not assist amplification of any definite bands and primers No. 24, 25 did not produce any scoreable polymorphic bands. In all the 25 selected primers amplified a total of 275 amplicons from 16 barley genotypes were detected, among them, 65 fragments (23.6%) were monomorphic and the rest (76.4%) were polymorphic between one or more genotypes. The cultivar genotypes were clustered according to their simple matching coefficient and complete-link methods. The least similarity was observed between wild genotype (Plot 21) and Valfajr. In general, Rihane with the average of 50.6% and Kavir with the mean of 60.8% had the least and the most similarity with other genotypes, respectively. Primer No. 27 amplified a 600 base pair fragment characteristic of tolerant cultivar lines and absent in susceptible genotypes. Therefore, it is likely that this DNA band be associated with drought tolerance. Clustering on the basis of 51% similarity ranked genotypes into 5 groups. The resulting dendrogram indicated that the cross of tolerant wild genotype (Plot 21) and susceptible Sina genotype which have low genetic similarity, also grouped in distinct cluster. This may be suggested as the most suitable cross to analyze QTLs (Quantitative Trait Loci) involved in drought tolerance.