The genetic diversity analysis of 90 barley samples, including 45 wild close relatives of barley from the Tibet region of China and 45 wild accessions from different countries throughout the Middle East, were carried out using ISSR and SSR markers. The results showed that Tibetan wild close relatives of barley had a higher genetic diversity than those from the Middle East. Ten ISSR primers amplified 91 allelic variants, of which 79 were polymorphic (86.81%), in the Tibetan genotypes and 82 allelic variants, of which 66 were polymorphic (80.49%), in the Middle East genotypes. Eleven primer pairs of SSR markers amplified 100 allelic variants among the Tibetan genotypes with 100 polymorphic bands (100%). Among the Middle East genotypes, 78 allelic variants were produced containing 77 polymorphic bands (98.72%). Moreover, the total gene diversity analysis (HT) values of Tibetan barley (0.227 for ISSRs and 0.126 for SSRs) were higher than those of Middle East (0.212 for ISSRs and 0.102 for SSRs). Cluster analysis of the ISSR and SSR results by the UPGMA method revealed two distinct groups correlated with the geographic origin of sampling. These results offer a new evidence for the theory of the origin of Hordeum vulgare L. To cite this article: A. Wang et al., C. R. Biologies 332 (2009).