In order to provide new evidence for the study of origin and evolution of flue-cured tobacco, genetic diversity and genetic relationships among 22 varieties of flue-cured tobacco were investigated with the technique of RAPD and Morphological Markers. RAPD Cluster analysis was carried out using software Bio ID++. A dendrogram was constructed by using the RAPD data and the integrated data, respectively. According to the dendrogram the varieties could be clustered into three groups at the level of D=0.30 by the first classification. At the level of D=0.23 by the second classification, the varieties could be clustered into 6 groups（Yunyan series and their genetic parent and Tezi 401 and its family progenies,；Majority varieties from American; 9804, Yanyan 97 and Coker176; CF965 and K07; Hongda; C2）. The results of two types of cluster analysis showed somewhat geographical origin and genealogical origin of the variety characteristics.