Satureja mutica (Lamiaceae) is an herbaceous medicinal plant which grows in Iran. The objective of the study was to obtain an overview of the genetic relatedness among and within seven populations of this species using inter-simple sequence repeat (ISSR). Fourteen ISSR primers amplified a total of 197 DNA fragments of which 176 (88.91%) were polymorphic. All ISSR primers were highly effective in discriminating among the populations. Genetic similarity coefficients ranged from 0.45 to 0.94, indicating considerable distance and diversity in the germplasm and were confirmed by clustering analysis. The dendrogram showed a clear clustering pattern of plants indicating a significant association between genetic similarity and geographical distance. Analysis of molecular variance revealed that a greater proportion of total genetic variation existed within populations (75%) rather than among populations (25%). The study indicated that ISSR markers were effective and reliable for assessing the degree of genetic variation of S. mutica. These findings can support future research on the selection of S. mutica for breeding and medicinal plant development.