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Identification and application of genetic diversity are essential to breeding programs success. In this study, genetic diversity of 20 rainfed barley genotypes were assessed using morphological traits as well RAPD and intron-exon splice junction (ISJ), semi-random markers. Results of this study showed that there were significant differences among genotypes for all traits, indicating high genetic variation among them. The highest and lowest broad sense heritability was related to spike length and grain yield, respectively The estimates of genotypic coefficient of variation (GCV) and phenotypic coefficient of variation (PCV) were high for number of grain per spike, and low for 1000-kernel weight, respectively. Mean of polymorphic percentage in ISJ marker was higher than RAPD marker. Cluster analysis showed that the distinctions based on morphological traits did not correspond with the distinction based on molecular data.The results showed that RAPD and ISJ markers were able to distinct two and six-rowed and also hulless and hulled barley genotypes. Distinction of three clusters did not follow the same pattern.There was significant and negative correlation between similarity matrices of molecular data and morphological traits, but similarity matrices of two molecular markers was significantly and positively correlated.