Abstract: In order to enrich the molecular markers of tobacco, and to speed up its genetic diversity analysis and utilization, 14 tobacco cultivars which had more genetic diversity were used for the screening of 22 pairs of SSR primers from chili or tomato. Eight pairs of primers were selected for their repeatability and polymorphic results, and they could generate 98 polymorphic fragments in other 24 tobacco samples. The primer polymorphic information was 0.451-1 with the average of 0.646. The Diversity coefficient of samples was between 0.3445-0.8626. The 24 of the samples could be identified and discriminated efficiently by primer 9. They could be classified into 4 groups at the level of GD 0.62, which was consistent with genetic relatives. Therefore solanaceae SSR primers were efficient in the study of genetic relationship, diversity and classification of variety resources with tobacco.