Abstract: The stability and consistency of tobacco leaf raw materials are key for ensuring the high-quality development of tobacco industrial companies’ products. Effective differentiation and identification of tobacco leaf raw materials is an urgent challenge that industrial companies need to solve. In this study, 23 flue-cured tobacco varieties mainly planted in the production areas of Tobacco Jiangsu Industrial Corporation were used as materials. Eventually, 12 pairs of primers with high polymorphism and clear bands were selected from 212 pairs of SSR primers by polyacrylamide gel electrophoresis detection. The number of alleles ranged from 2 to 5, and the polymorphism information content ranged from 0.333 8 to 0.571 1. Cluster analysis revealed that each variety was clustered into four categories based on genetic background. Based on the population genetic structure and principal component analysis the tested materials were divided into two subgroups. The distribution results of varieties within the groups were highly consistent, and were largely agreed with the pedigree relationship. This set of primers was highly efficient and reliable, and it could be used for constructing DNA fingerprints and genetic diversity analysis of flue-cured tobacco varieties. The results from this study could provide theoretical basis for the breeding utilization and identification of tobacco leaf materials of main flue-cured tobacco varieties in the Tobacco Jiangsu Industrial Corporation core production areas.