The genetic diversity of 21 populations of 3 varieties of KengyiliaYen was investigated by EST-SSR markers. There are 103 polymorphic bands were created by 60 avaliableEST-SSR primers which were selected from 97 primers in those materials. Every primers amplifying 1 to 5 bands ,and every band have an average number of alleles of 1.70. The genetic diversity of Kengyilia thoroldiana was the highest and that Kengyilia hirsute was the second highest. Kengyilia grandiglumis with the lowest PP of 44.66% in genetic diversity. The distance between Kengyilia thoroldiana and Kengyilia grandiglumis of genetic similarity analysis was minimal and the genetic similarity was maximum. The genetic distance of Kengyilia hirsute and Kengyilia grandiglumis and Kengyilia thoroldiana was far. The number 1 population of Kengyilia thoroldiana was far compare with other populations in genetic distance. The number 14 population of Kengyilia hirsute was far compare with other populations in genetic distance. The number 18 population of Kengyilia grandiglumis was far compare with other populations in genetic distance.The 21 populations was classified 3 groups :10 Kengyilia thoroldiana populations,7 Kengyilia hirsute populations and 4 Kengyilia grandiglumis populations by NTSYS 2.10e. Resonable reference can be provided to further research of genetic resource in Triticeae in this research.