Chrysanthemum (Chrysanthemum morifolium) is one of the most important ornamental plants which plays a significant role in the development of gardening industry in the world. The knowledge of genetic diversity is one of the prerequisite criteria for Chrysanthemum breeding with important economic goals. Molecular markers have a significant share in elucidation of inter and intra species genetic diversity. To this end, genetic diversity of a number of Iranian cultivars was molecularly investigated by sequencing a part of rDNA, using ITS4 and ITS5 primers. Genetic distance between Chrysanthemum cultivars ranged from 0.05 to 10.15, demonstrating the power of ITS region in revealing the genetic diversity among cultivars of morifolium, suggesting Iranian cultivars have been genetically improved from morifolium species. Genetic diversity assessment of Iranian Chrysanthemum cultivars demonstrated that presumably inter, intra species or even inter population hybridization may have been involved in creating enormous genetic diversity among Chrysanthemum cultivars.