In order to detect the resistance genes of germplasm resources against barley stripe disease and understand their genetic diversity, in this study 91 barley accessions were inoculated with barley stripe disease pathogen by sandwich method, followed by the genetic diversity analysis using 31 polymorphic SSR markers. Of them, four accessions were identified to be complete resistance (free of diseased symptom), 6 highly resistant, 33 resistant, 42 susceptible and 6 high susceptible. Genotyping by 31 SSR markers disclosed 238 alleles, with a mean of 7.677 alleles in each of markers and the range of 2 to 19 alleles. The frequency of major loci ranged from 0.236 to 0.951, with an average of 0.394. The gene diversity index ranged from 0.094 to 0.871, with an average of 0.667. PIC ranged from 0.091 to 0.860, with an average of 0.613, while GS ranged from 0.103 to 1.000, with an average of 0.522. This collection were divided into three groups at the genetic similarity coefficient level of 0.783, each containing 86, 2 and 3 accessions, respectively. The population structure analysis suggested three groups, each including 47, 33 and 32 materials, respectively. 97.80% of 91 genotypes had Q value greater than 0.6. Gained from disease resistance identification and genotyping, this study provided reference for selecting parental lines in breeding of barley leaf stripe resistant varieties.