Introduction: Zataria multiflora is an important medicinal plant of the Lamiaceae family in Central and Southern Iran. This plant is at the risk of extinction as a result of wasteful harvesting due to growing demand and high economic value. Materials and Methods: In this study, the genetic diversity of 25 different accessions of Z. multiflora, collected from provinces including Hormozgan, Fars, Sistan and Balouchestan, Bushehr, Yazd, Kerman and Isfahan were examined using Inter Simple Sequence Repeat (ISSR) markers. To extract the DNA, five samples taken from the leaves of each accession were transformed into bulks; then, their concentrations were homogenized and 15 primers were used for the remainder of the experiment. Results: The primers produced 83 polymorphic strands altogether, with an average polymorphism percentage of 77.30%; meanwhile, the highest polymorphism percentage (100%) was achieved via primers including 816D, 824H, 836P, and 844S. The lowest polymorphism percentage was obtained from 811C and 834N primers. The results obtained from Jaccard similarity coefficient in the NTYSIS software showed that the genetic similarity of Z. multiflora accessions varies between 0.32-0.82. The lowest similarities were observed in accessions taken from Fanuj and Ashar, Mehriz, and NasrAbad, and two accessions of Mehriz and Khafr. However, the highest similarities were seen among accessions of NalShah GhandAab and Kerman. In principal component analysis, three of the first components explained 40.44% of changes in the entire data. Following a cluster analysis based on UPGMA algorithm, accessions were classified into six groups. Conclusion: It can be concluded that the ISSR markers are suitable for examining the genetic diversity of Z. multiflora accessions.
