Ziziphora tenuior L. is a medicinal plant species of the genus Ziziphora (Labiatae) that grows in different areas of Iran. In order to study the population genetic structure in Ziziphora tenuior,we collected 107 plant specimens from 20 geographical populations that are located in 17 provinces. ISSR molecular markers were used for genetic diversity analysis. The populations studied revealed intra- and inter-population genetic variability. AMOVA test showed significant genetic difference among the studied populations. STRUCTURE plot identified two main gene pools for Ziziphora tenuior in Iran. These populations showed isolation by distance and restrict gene flow occurred among them.