Barley is an important cereal crop cultivated in many parts of the world since ancient times. Genetic diversity was studied in a set of 16 barley (Hordeum spp) genotypes using 25 microsatellite (SSR) loci. A total of 94 different alleles distributed over all the seven chromosomes of barley. Out of these, 76 alleles were polymorphic with an average of 3.04 alleles/primer. Major allele frequency, gene diversity and PIC were estimated based on allele frequencies, having the mean values of 0.543, 0.571 and 0.513, respectively. Principal component analysis indicated the presence of high genetic variability among barley genotypes. ATACO, Canela, Local Abiad Sulaimani and Amal2 were identified to be highly distanced from other genotypes. The dendrogram created by hierarchical cluster analysis using the UPGMA algorithm was able to discriminate the genotypes into 7 groups according to their genetic distance. Both analyses parallel to principal coordinate analysis are indicating the close relatedness between Quinn, Zanbaka, Al-khair and Arta/3/Avar genotypes that exchange genetic material through continuous previous breeding programs. These values indicate high differentiation ability of SSR markers for genetic studies in barley and their utility for barley genetics and breeding.