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Water stress is one of the most common environmental stresses that limited tobacco production in IRAN. Identification of genetic diversity in tobacco germplasm and genetic relationships between traits help to improve tolerant varieties. In this study, the genetic diversity of 100 flue–cured tobacco (Nicotiana tabacum L.) cultivars was analyzed using 15 morpho-physiological traits and 25 ISSR primers. The cultivars were cultured in a simple lattice design (10×10) with two replications (with and without water stress) in Tirtash Tobacco Institute, Iran. Results showed relative water content (RWC) and cell membrane stability (CMS) in normal condition were significantly higher than stress condition. The estimated broadsense heritability was low for RWC and CMS that represents large environmental effects on these two traits. The results of genotypes clustering by UPGMA method with ISSR markers and by WARD method with morph-physiological traits did not match. Primer UBC814: (CT) 8A with 16 polymorphic bands of the 17 bands, had higher resolution than other primers and seems appropriate for molecular diversity studies in tobacco. The K394 genotype was identified as well as drought tolerant varieties. We can use results of this study for selecting genotypes with great genetic differences and chose the desirable traits and use in breeding programs for producing high heterosis hybrid with tolerance to drought stress in tobacco.