Abstract: SSR marker is the ideal techniques for analysis of plant genetic diversity. In this study the genetic diversity among 13 main cultivars in Yunnan province were measured using these tobacco SSR markers which published recently. 92 SSR located on 24 tobacco linkage groups were screened, among which 20 were polymorphic. Fifty-two alleles were amplified in 13 cultivars using these 20 SSR primer pairs, with 2.6 alleles per primer pair. The genetic distance (GD) in 13 tobacco cultivars was calculated based on the SSR marker polymorphism, ranging from 0.0090-0.4286, with the average of 0.2237. This result indicates the genetic variation of these cultivars were very little. Tobacco SSR markers can be used to analyze effectively the genetic diversity of tobacco germplasms and of great value for molecular genetic mapping and marker assisted selection in tobacco breeding. Key words: tobacco cultivars SSR markers Genetic diversity.