Abstract： In order to enhance the collection，conservation and utilization of yellow sun-cured tobacco resource, 32 yellow sun-cured tobacco resource were analyzed by 106 pairs of microsatellite(SSR) markers screened. On the base of the different origin, these tobacco resource were clustered into three populations (Z was from tobacco mid-genebank. G was from Hezhou of Guangxi province, M was from Baise of Guangxi province).The results were as follows:1) Totally 110 loci were produced with 85 polymorphic loci (77.27%) using106 pairs of SSR selected from 740 primer combinations. The range of variant coefficient was 0.49-0.95 based on molecular cluster. The results showed that the genetic basis of yellow sun-cured tobacco resource was narrow.2) The order of three populations’ diversity was Z>M>G. The genetic coefficient was 0.7762 between Z and M, in addition, the genetic coefficient was 0.8963 between G and M.3) 32 tobacco resource were divided into 2 groups at coefficient 0.49. The first group included Cunsanpi and Daninghanyanzi germplasm. The second group included 3 sub-groups. Gonghuishaiyan from Z population was clustered with the new collections, and other tobacco resource from Z population wasn’t clustered with the new collections.4) The genetic distance of the homonymic resource named Daningyan from different origins was far, and the coefficient was 0.788. The genetic distance between Gonghuibaogaiyan and Fenghuanghuangyan was nearest, and the coefficient was 0.939.