Naked barley is widely planted in Qinghai-Tibet Plateau in China and essential for the Tibetan life in those regions. In this study, the genetic diversity of 64 cultivated naked barley accessions from Qinghai-Tibet Plateau in China was analyzed using 30 mapped SSRs linked with the important traits of barley improvement. A total of 132 alleles were identified at 22 polymorphic SSR loci, with the number of each locus ranging from 2 to 15, the polymorphism information content (PIC) values ranging from 0.16 to 0.91, and with an average of 0.65. Of the selected SSRs, 13 SSR markers with high PIC value were highly efficient for the genetic analysis of Chinese barley. The accessions were divided into five main groups by cluster analysis and could be differentiated from each other. The genetic diversity in the Tibet accessions was slightly higher than in the Sichuan accessions. It is found that there were specific genes linked with the collecting sites. These results indicate the cultivated naked barley from Qinghai-Tibet Plateau in China are highly polymorphic and could be considered as an important resource bank for cultivated naked barley breeding in the future.