Sweet flag (Acorus calamus) is a perennial, semi-aquatic and aromatic plant of the family Acoraceae that, in addition to its multiple medicinal properties, is used in health, food and agricultural industries (as pest control). This research was conducted to comparasion study of genetic diversity of natural and regenerated plants from tissue culture of Arzefon, Pelesk and Alandan populations of Sweet flag by using ISSR molecular markers. Out of 15 screened primers, 9 primers produced the most polymorphic bands. Totally, these primers generated 83 bands, of which 52 bands (62.65%) were polymorphic. The percentage of polymorphic locus for natural and regenerated plants was 43.37% and 55.42%, respectively, and Nei’s genetic diversity (H) was calculated to be 0.239 for the two studied groups. The Shannon’s index (I) for natural and regenerated plants was estimated to be 0.251 ± 0.033 and 0.299 ± 0.031, respectively. Among the natural and regenerated groups, the highest genetic similarity was observed between the samples of Alandan population (0.63), and the lowest value was observed between the samples of Pelesk population (0.44). Analysis of molecular variance (AMOVA) showed that 94 % of genetic variation attributed to whithin groups and 6 % to between groups. Based on the results, the genetic diversity of the regenerated plants was higher than the natural plants. According to the results of the present research, the lowest rate of genetic divergence was observed between natural and regenerated plants of Alandan populatiuon, so the plants of this population could be suitable for domestication and cultivation in Iran.