Abstract: The genetic distance of sixty-nine flue-cured tobacco germplasm resources were analyzed by the field performance characters and SSR markers. The genetic distance was 0.32-7.83 among 69 flue-cured tobacco germplasm varieties, with an average of 2.80. The genetic similarity index was 0.30-0.96 among 69 flue-cured tobacco varieties, with an average of 0.70. Cluster analysis based on the matrix of genetic distance and genetic similarity index, respectively, was performed. Clustering results showed that the tested tobacco varieties could be divided into foreign varieties, domestic varieties, early-flowering type, late-flowering type etc.. The combination of morphological markers and molecular markers to evaluate the genetic diversity of flue-cured tobacco germplasm resources may reveal the interspecific genetic diversity and genetic relationship of tobacco, providing the theory basis for parental selection in cross breeding of tobacco genetic breeding.