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Tibetan annual wild barley is rich in genetic variation. This study was aimed at the exploitation of new SSRs for the genetic diversity and phylogenetic analysis of wild barley by data mining. We developed 49 novel EST-SSRs and confirmed 20 genomic SSRs for 80 Tibetan annual wild barley and 16 cultivated barley accessions. A total of 213 alleles were generated from 69 loci with an average of 3.14 alleles per locus. The trimeric repeats were the most abundant motifs (40.82%) among the EST-SSRs, while the majority of the genomic SSRs were di-nuleotide repeats. The polymorphic information content (PIC) ranged from 0.08 to 0.75 with a mean of 0.46. Besides this, the expected heterozygosity (He) ranged from 0.0854 to 0.7842 with an average of 0.5279. Overall, the polymorphism of genomic SSRs was higher than that of EST-SSRs. Furthermore, the number of alleles and the PIC of wild barley were both higher than that of cultivated barley, being 3.12 vs 2.59 and 0.44 vs 0.37. Indicating more polymorphism existed in the Tibetan wild barley than in cultivated barley. The 96 accessions were divided into eight subpopulations based on 69 SSR markers, and the cultivated genotypes can be clearly separated from wild barleys. A total of 47 SSR-containing EST unigenes showed significant similarities to the known genes. These EST-SSR markers have potential for application in germplasm appraisal, genetic diversity and population structure analysis, facilitating marker-assisted breeding and crop improvement in barley. [ABSTRACT FROM AUTHOR]