Abstract: Genetic diversity was assessed among 24 flue-cured tobacco varieties by ISSR (inter simple sequence repeats). A total of 100 ISSR primers were used to amplify the DNA from these varieties, of which 10 primers produced reproducible amplified products. Using polyacrylamide gel electrophoresis 208 bands were identified, of which 141 bands were polymorphic among the flue-cured tobacco varieties analyzed. Each primer produced 7~37 bands, the length of which ranged 200~2 400 bp. The ratio of polymorphic bands (PPB) was 67.79%. By cluster analysis based on ISSR markers using UPGMA, 24 varieties were divided into 5 major groups, in which the biggest group consisted of 12 varieties derived from Coker319. The genetic similarity index was 0.66~0.85 among 24 flue-cured tobacco varieties. Low genetic diversity among flue-cured tobacco varieties suggested that it is necessary to expand the genetic base of the flue-cured tobacco. 24 varieties could be distinguished by using 2 ISSR markers. The result also indicated that ISSR analysis was suitable for varietal identification and the study on genetic diversity of tobacco germplasm.