To study the genetic regularity of tobacco black shank resistance of different parents, and to identify the genomic locations contributing to the resistance, 220 F2 populations were constructed from a cross between Beinhart1000-1 and susceptible variety, xiaohuangjin1025. Through disease inoculation and genetic analysis, we found that the resistance to black shank of Beinhart1000-1 was controlled by the dominance genes. 70 stable SSR primers were used for QTL mapping of resistance to black shank. We got 14 linkage groups which included 52 SSR markers. Five QTLs are discovered on No.2, No.3, No.3, No.6 and No.12 genetic linkage groups, and account for 6.2%, 6.0%, 6.7%, 5.6% and 5.1% of the phenotypic variance, respectively. In order to further study the genetic regularity of tobacco black shank resistance of different parents, and to posit the resistance gene loci, the 5 SSR markers has certain value in marker-assisted breeding. These results could facilitate our understanding of the inheritance of resistance to black shank in tobacco by marker-assisted selection.