Chrysanthemum is an ornamental-medicinal species with great economic value. Evaluation of genetic diversity of chrysanthemum cultivars has been critical to the effective management of breeding programs and the conservation of genetic resources. In this study, 13 ISSR primers were used to investigate the genetic diversity of 30 different chrysanthemum cultivars. The results of this study showed that of 86 bands that were produced, all bands were polymorphic, so that the number of polymorphic bands varied from 5 to 9 per primer. Primers marking index (MI) in cultivars ranged from 1.40 to 4.32 and the polymorphic information content (PIC) content of primers ranged from 0.28 to 0.48 in each primer fluctuated. The results of principal coordinate analysis showed that the first three components were able to explain 29.27% of the total variance. Also, genetic relationships between samples were calculated using Jaccard similarity coefficient and cluster analysis by UPGMA method divided 30 cultivars into six main groups. The less genetic distance 0.38 was observed among the cultivars of "Fariborz" and "Farahnaz" by a similarity of 0.38, and the highest genetic distance was observed among the cultivars of "Oran"' and "Taban3"' by a similarity of 0.91. Primers T (GA) 9 and (AGC) 5GA had the highest values of molecular markers that can be introduced as useful and desirable primers for differentiation of chrysanthemum cultivars.