Barley ranks below wheat, corn and rice in total world production. Barley is one of the most important crops in Iran. In this study, genetic diversity of 20 rainfed barley genotypes were assessed using morphological traits as well as 20 primers of ISJ semi-random markers. There were significant differences among genotypes for the all traits, indicating high genetic variation among barley germplasm. Based on molecular data, 133 bands were detected and 89 bands were polymorph. The mean number of bands was 6.65 per primer. According to cluster analysis of similarity matrix of molecular and Euclidean distances of morphological data, similarities ranged between 0.42-0.85 and 1.44-43.22, respectively. Based on molecular data and morphological traits, the highest similarities were belonged to genotypes number 2, 8 (0.85), and 5, 8 (1.44) respectively. The results showed that intron-exon splice junction (ISJ) markers and morphological traits rather could distinct two and six-rowed and also hulless and hulled barley genotypes. Distinction of two clusters did not follow the same pattern.