Artemisia is one of the most diverse genera in the Asteraceae family. The genus is wildly distributed in Irano-Turanian habitats and includes 34 species in Iran. Here, for the first time the essential oil variability, antioxidants and anti-cholinesterase and anti-tyrosinase activities of extracts of three Artemisia species (A. tournefortiana, A. khorassanica, A. haussknechtii), from different regions of Iran were evaluated. Based on GC–MS analyses, 81.84% to 98.70% of the total oils were identified. Cluster analysis grouped the studied populations in three different chemotypes. The highest and the lowest essential oil contents were observed in A. khorassanica and A. haussknechtii species, respectively. Camphor, en-in-dicycloether, 1,8-cineole and (Z)-β-farnesene were the dominant components of essential oil in investigated ecotypes. The results revealed that the total phenol content was higher in A. tournefortiana collected from Kerman and A. haussknechtii collected from Chaharmahal and Bakhtiari. However, the lowest phenol content was recorded for A. haussknechtii collected from Isfahan province. The highest flavonoids content was found in A. tournefortiana collected from West Azerbaijan and A. khorassanica collected from North Khorasan. The highest FRAP antioxidant activity was observed in A. tournefortiana (Kerman) and the lower amount was in A. haussknechtii collected from Kohgiluyeh and Boyer-Ahmad. The highest antioxidant activity by DPPH method was in A. khorassanica collected from South Khorasan and the lowest activity was in Isfahan's A. haussknechtii. The acetycholine esterase inhibitory activity was higher in A. tournefortiana collected from West Azerbaijan; and the lowest activity was in A. haussknechtii collected from Chaharmahal and Bakhtiari province. The highest tyrosinase inhibitory activity was in A. khorassanica collected from North Khorasan; and the lowest was in A. haussknechtii collected from Chaharmahal and Bakhtiari.