Abstract: Vitex pseudo-negundo (Hausskn.) is a perennial plant of Lamiaceae family which grows naturally in the vicinity of seasonal rivers of Iran. The present study was aimed to characterize the essential oils composition of five Iranian populations of V. pseudo-negundo. The aerial parts of the plant were collected at flowering stage. GC-FID and GC-MS analyses of the essential oils identified a total of forty-one components in the examined essential oils, accounting for 95.8- 98.5 % of the total composition. According to the results, no significant differences was observed among the main compositions of the essential oils. In all investigated populations, the main fraction was represented by monoterpene hydrocarbons with α-pinene (25.3-29.2 %) and limonene (7.89-9.85 %) as the most abundant components. (E)-β-methyl ionone (12.38-17.37 %), (E)-β-caryophyllene (3.7-9.8 %) and β- eudesmol (5.0-6.1 %) were the other main components of the essential oils.