The research aimed to evaluate the genetic diversity and the genetic relationship between 16 Hordeum vulgare genotypes (variaties and new lines), using SSR markers. 32 primer pairs were used in the analysis, five of them amplified primers monomorphic alleles, while the remaining primers detected different alleles. A total of 126 polymorphic alleles were revealed by the 27 primer pairs. The number of different alleles detected on alocus ranged from 2 to 11with a mean of 4.66 alleles per locus. The polymorphic information content (PIC) values ranged from 0.21 to 0.88 with a mean value of 0.49, and the gene diversity ranged from 0.23 to 0.89 with a mean value of 0.53.The dendrogram was established using SSR data clustered into three major groups. The smallest genetic relationship was observed between line39 and line38 . The results proved the efficiency of SSR markers in the estimation of genetic diversity and in the clustering of the barley genotypes in Syria.