Characterization of genetic diversity has long been a major goal in tobacco breeding programs. Information on genetic diversity is essential for a rational use of genetic resources. In the present study, the genetic variation among 72 flue-cured tobacco genotypes was evaluated using microsatellite markers (SSRs). A set of 104 alleles was generated at 30 SSR loci. The mean number of alleles per locus (n a ) and the effective allele number (n e ) were 3.467 and 2.358, respectively. The expected heterozygosity ranged from 0.29 to 0.75 with average of 0.54. Several methods were used to construct the similarity matrices and dendrograms. The co-phenetic correlation coefficient, which is a measure of the correlation between the similarities represented on the dendrograms and the actual degree of similarity, was calculated for each dendrogram. Among the different methods, the highest value (r=0.76368) was observed for the UPGMA created based on Jaccard's similarity coefficients. The genetic similarity among the tobacco genotypes calculated by using Jaccard's similarity coefficient ranged from 0.08 to 0.84, suggesting the presence of high molecular genetic variability among the studied tobacco genotypes. Based on UPGMA clustering method all studied flue-cured tobacco genotypes, except for 'Glustinusa Rasht' , were placed in three distinct groups. We observed an obvious heterotic pattern in the studied flue-cured germplasm corresponding to genetic distances and classification dendrogram, which persuades exploitation of heterosis in flue-cured tobaccos.