Abstract: In order to accurately evaluate the genetic diversity of introduced tobacco germplasm resources, 38 tobacco germplasm resources were analyzed with 42 SSR primers with good polymorphism. A total of 22 pairs of polymorphic primers were selected based on higher identification ability. The average number of alleles was 3.68. The average diversity index of Nei was 0.451, and the average Shannon information index was 0.789. The UPGMA cluster analysis showed the specificity of Honghuadajinyuan. The genetic structure analysis showed that these 38 accessions were classified into two groups (group 1 and group 2), which included 16 and 22 accessions, respectively. The range of variation of group 1 was larger than that of group 2. Most accessions in group 2 showed less than 210 cm in plant height, more than 20 in the number of leaves, 10-11 cm in the stem circumference. Furmore,the accessions in group 2 were susceptible to brown spot disease but resistant to bacterial wilt. The results can provide theoretical basis for improving SSR molecular marker screening efficiency and innovative utilization of some flue-cured tobacco germplasm.