Assessment of the extent of genetic variability within a cultivated crop has important consequences in plant breeding and conservation of genetic resources. Random amplified polymorphic DNA (RAPD) markers were used to produce barley genomic DNA fingerprints. The aim of this work was to investigate the efficacy of polymorphism contained in such fingerprints on disclosing genetic relationships among 26 barley genotypes including advanced lines and released cultivars. Among the 21 primers the percentage of polymorphic bands ranged from 33% (OPG-07) to 83% (OPG-02), showing a high degree of molecular variation. The highest genetic similarity was observed between genotypes Karan-521 and Karan-741(58%), and the lowest genetic similarity was observed between the cultivars Lakhan and C 138 (3.7%). The similarity matrix by the UPGMA algorithm delineated the above genotypes into four major clusters. The results demonstrate the utility of RAPD markers in differentiating Indian barley varieties. KT and SJY were supported by 2008 National Agricultural Germplasm Management System Establishment Program.