Dragonhead (Dracocephalum moldavica L.), an endangered herb, faces significant threats due to the loss of biodiversity driven by land-use change. The seeds of seven landraces of D. moldavica were collected from various native populations in different regions of Northwest Iran. Subsequently, they were cultivated in 10 replications, spanning a total of 70 plots. This cultivation took place at the Ekbatan station of the Agriculture and Natural Resources Research Center in Hamadan province. This study aimed to assess the morphological traits, essential oil (EO) content, and genetic diversity within seven populations of Dracocephalum moldavica. Significant variations in morphological traits were observed among populations, indicating phenotypic diversity and potential for selective breeding programs. The EO content ranged from 0.03 % to 0.13 % (w/w), with the highest in the HO population and the lowest in the KE population. EO yield varied from 1.97 in the KE populations to 13.97 Kg/ha in the HO populations. The chemical composition of the EOs varied among populations and consisted of 18–24 components, with geranyl acetate being the dominant component. PCA analysis identified population differences primarily related to geranyl acetate, nerol, geraniol, neryl acetate, and neral. The observed variation in EO composition may result from genetic and environmental factors. ISSR analysis revealed high genetic diversity, with 84 out of 94 scorable bands being polymorphic. Cluster and PCoA analysis revealed distinct genetic clusters among populations, although some diversities did not align with geographical origin, suggesting seed transfer or high heterozygosity. The study highlights the effectiveness of ISSR markers in assessing genetic diversity and provides insights into the genetic structure and variability of D. moldavica populations. Furthermore, the correlation between ISSR markers and phytochemical traits suggests their potential for breeding programs to enhance important traits in D. moldavica. Considering both phytochemical and morphological properties is crucial in breeding programs due to pleiotropic effects of genetic loci.