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Control of Cucumber mosaic virus (CMV) andTobacco etch virus is difficult (TEV) mainly because of the lack of resistant tobacco varieties. In this study, the resistance to CMV and TEV of 22 tobacco cultivars, grown under both greenhouse and field conditions, was evaluated from 2007 to 2012. The genetic relationships among these cultivars were also assessed using ISSR methods. Based on the disease index and DAS-ELISA results, one cultivar (Coker86) was characterized as resistant to CMV, and one cultivar (Shuangkang70) was characterized as resistant to TEV. A total of 145 repeatable amplified bands were generated with six ISSR primers, of which 139 were polymorphic. The tobacco cultivars were grouped into five major clusters according to phylogenetic analysis; the similarity coefficient between cultivars ranged from 0.476 to 0.855. This study provides an overall assessment of tobacco cultivars and the results should prove useful for both the control of tobacco virus diseases at present and the improvement of tobacco varieties in future breeding projects.