Helichrysum (family Asteraceae) is a large, heterogeneous and polyphyletic genus. Most of the Helichrysum species have ornamental and medicinal values. Helicrysum leucocephalum Boiss., has wide geographical distribution in Iran and forms several local populations. The present investigation was performed to study inter populations, genetic, cytogenetic and morphological diversity of this medicinal species. The AMOVA test showed significant genetic difference among the studied populations but the Mantel test did not show correlation between the genetic distance and geographical distance of these populations. STRUCTURE and reticulation analyses showed some degree of genetic admixture and gene exchange among the studied populations. These populations had different chromosome numbers and also differed significantly in the size of their chromosomes.