Fifty-two SSR markers were used to evaluate the genetic diversity of 33 Qinghai-Tibetan wild barley accessions, 56 landraces collected primarily from other parts of China, and 1 Israeli wild barley accession. At the 52 SSR loci, 206 alleles were detected for the 90 accessions, among which 111 were common alleles. The number of alleles per locus ranged from 1 to 9, with an average of 4.0. Polymorphism information content (PIC) values ranged from 0 to 0.856 among all the markers, with an average of 0.547. The PIC value of Qinghai-Tibetan wild barley varied from 0 to 0.813 with an average of 0.543, while in landraces, the markers showed a range of 0 to 0.790 with an average of 0.490. The SSR markers could clearly differentiate the Qinghai-Tibetan wild barley from the landraces. Twenty-four unique alleles were observed in Qinghai-Tibetan wild barley, and the frequency of unique alleles in Qinghai-Tibetan wild barley was about 2.1 times higher than that in the landraces, on average. Five of the 7 chromosomes had more unique alleles in the Qinghai-Tibetan wild barley, but chromosome 2H had more unique alleles in the landraces. The presence of many unique alleles may reflect the adaptation of this barley germplasm to diverse environments and production systems.