106 accessions of Tibetan wild barley, including 50 accessions of the two-rowed wild barley Hordeum vulgare ssp. spontaneum(HS), 27 accessions of the six-rowed bottle-shaped wild barley H. lagunculiforme(HL) and 29 accessions of the six-rowed wild barley H. agriocrithon(HA) that separately represent different agrigeographical regions of Tibet, were used to study the genetic diversity and genetic differentiation using SSR markers selected from seven barley linkage groups. 229 allelic variants were identified with an average of 7.6 alleles/locus. The average of total number of alleles per locus in HA(6.4) is much higher than that in HS(3.9) and HL(3.4). The genetic diversity and its standard deviation among the three subspecies were in the order of HS>HL>HA. Very significant genetic differentiation was observed among the three subspecies of wild barley. Comparisons of the results from this and previous studies showed a strong Oriental-Occidental differentiation of barley, and that Shannan region of Tibet might be the center of origin of the Tibetan two-rowed wild barley, thus supporting not only the hypothesis of a mono-phyletic origin of cultivated barley but also the proposition that the Tibetan two-rowed wild barley as ultimate progenitor of Chinese cultivated barley.