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Determination of genetic diversity of breeding material is the first step in breeding programs. Evaluation of tobacco genetic diversity is essential for breeding programs and preservation of genetic resources. Genetic diversity level in tobacco genotypes, is very important for selection of parents in breeding programs. In this study, genetic diversity of 100 tobacco genotypes was evaluated using 25 ISSR markers. Banding pattern based on the presence or absence of the bands showed with 0 and 1, respectively. Out of 237 fragments produced in total cultivars, 195 bands were polymorphic and average of polymorphism ranged from 4 to 12 per primer. Average of polymorphism percentage was 94.10. To determine the efficiency of ISSR markers, PIC and their polymorphic percentage was calculated. UBC 818, UBC 812 and UBC 815 had the best marker parameters and were introduced as the best primers for assessment of genetic diversity. In order to evaluate the genetic similarity between cultivars, different similarity coefficient (SM, Dice and Jaccard) was calculated and Mantel corresponding test was performed. Finally, dendrogram was drawn based on SM similarity coefficient and UPGMA algorithm and the Cofenetic coefficient was calculated. All genotypes formed two distinct clusters indicating the high efficiency of used primers in amplification the approximate parts of the genome. The principle coordinate analysis showed that the first three components could explain 79.65 % of total variance. Totally, evaluation the tobacco genetic diversity using ISSR markers is suitable and ISSR marker can be used as appropriate marker system to identify the diversity and genetic relationship for breeding programs of this plant.