Betony (Stachys lavandulifolia) is one of the 34 wild species of important genus Stachys from Lamiaceae family which grows naturally in Iran [1]. In present study, seven endemic populations of S. lavandulifolia (including 56 samples) from five provinces were investigated. ISSRs (Inter simple sequence repeats) were used to survey genetic diversity and to infer population structures. In total, 12 used primers produced 176 fragments which all of them (100%) were polymorphic. By using NTSYS-pc software and UPGMA method, the samples were divided into six groups based on Jaccard genetic similarity matrix. Principal coordinates analysis (PCoA) confirmed the clustering results. POPGENE software was used to describe genetic variation within populations according to Shannon's index and Nei's gene diversity analysis. Genetic diversity within the populations of Azna and Toureh was the most and the least, respectively. The average observed number of allels to the effective number of allels (Na/Ne) was obtained to be 83% wich showed that the gene distribution were balanced in each population. Moreover, GeneAlex and POPGENE software’s were used to analyse genetic diversity and molecular variance among populations. Gst and Nm indexes average of the populations were accounted 0.20 and 1.94, respectively, indicating a balanced gene exchange among seven studied Betony populations. Molecular variance analysis of the populations (AMOVA) showed that genetic diversity within and among the populations were 75% and 25%, respectively. According to the results, Nei's genetic similarity ranged between 0.79-0.97, the most similarity was distinguished between Toureh with Zia-Abad and Khoramdareh whereas the least similarity was between Toureh and Salavat-Abad. STRUCTURE analysis revealed four subpopulation (K=4) in Betony populations. According to the results, ISSR marker could be used as a valuable tool for getting molecular information and investigating genetic diversity. In spite of overharve
