Abstract: To understand the genetic diversity of germplasm in the germplasm bank against black shank from the molecular level, and to afford theoretical and technical support for broadening the genetic diversity of germplasm against black shank and enhancing the utilization rate of the germplasm, genetic diversity of 73 resistant tobacco germplasm to black shank disease, including two Nicotiana tabacum population (flue-cured tobacco and sun-cured tobacco), came from national storage of tobacco germplsam was evaluated based on 34 selective polymorphic primer pairs. Results showed that 88 bands, all of which were polymorphic, were amplified using 34 SSR primers, with an average of 2.6 variable sites per primer pair. A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis (0.4200), Shannon’s information index analysis (0.6803). The percent of tobacco varieties, with a genetic similarity coefficients ranged from 0.5410 to 0.9818, was 63.57%. This indicated that there was a low genetic diversity among the resistant tobacco germplasm to black shank disease in China, and had no significant relationship whit its cultivated type. It had a significant meaning to widen the genetic base of the resistance tobacco varieties to black shank disease through exploring the applying of the wild tobacco.