Yarrow ( Achillea L.) is one of the youngest evolutionary genera of Asteraceae family which is scattered all around the world. More than 100 species have been recognized in this genus. To date, different medicinal applications of yarrow have made it known as an important medicinal plant. Nineteen species of Achillea have been recognized in Iran distributed in different geographical and ecological regions of the country. Among these species A. millefolium is widely distributed in North Hemisphere including Iran and has extensive application in pharmaceuticals. Populations of A. millefolium species in Iran are dispersed in North (N), West (W), North-West (NW), and West-Central (CW) based on Flora Iranica reports. Two major suecies, A. millefolium su. millefolium and A. millefolium su. elbursensis are recognized in the country with the latter being endemic to Alborz Mountain in Northern regions of Iran. Information on the levels of genetic variation within and among natural populations provide fundamental insights on the evolutionary processes and for the establishment of effective and efficient conservation strategy for small and endangered populations. In recent years, the use of molecular markers has become an important tool to study genetic diversity of various medicinal plants and endangered species. The use of molecular markers with morphological data can improve the classification and evolutionary studies in many plant species. Inter-simple sequence repeat (ISSR) markers are widely used in genetic diversity studies because they need no prior DNA sequence information, development costs are low, and laboratory procedures can easily be transferred to any plant species. In this study, forty accessions from eight populations of A. millefolium were collected from different geographical regions of Iran based on Flora Iranic. Sampling strategy was designed to cover the most distribution range of the species as widely as possible. In addition, 13 accessions from different countries in N hemisphere were included. ISSR and morphological markers were used for the detection of genetic polymorphism in this medicinal herb collected from various geographical regions of Iran and North Hemisphere. Fourteen primers were used to amplify 228 bands out of which 199 (87.28%) were polymorphic. Simple-Matching (SM) similarity indices were subjected to UPGMA cluster analysis. The dendrogram revealed three major groups. Group 1 included the Iranian genotypes (SW Asia), while group 2 was composed of European and American genotypes. The third group contained an accession from Japan (E Asia). Iranian accessions in group one further divided into three major sub-groups according to their geographical distribution. In this group, generally more genetic variation was observed in Northern and West-central populations than the ones from Western and North-Western