Plant responses to multiple environmental conditions in the habitat are characterized by intra-species variety. Ferula assa-foetida L. is a native medical plant in Iran, which has a wide range of therapeutic and pharmacological properties. The objective of the current study was to identify changes in phenolic content, antioxidant capacity and essential oil (EO) composition in different tissues (leaf, flower and fruit) of three populations of F. assa-foetida L. (Sarbisheh, Nehbandan, located in South Khorasan province of Iran). The results showed that total phenolic content (TPC), total flavonoid content (TFC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity in the leaves of Sarbisheh were higher than in other treatments. The amount of TPC, TFC and DPPH scavenging activity in leaves was higher than in other tissues in the Sarbisheh population, increasing by 31 and 70%, 41 and 20%, 8 and 13%, respectively, over that of flowers and fruits. The main EO compounds of leaves were α-pinene (11.86–13.56%), β-pinene (6.80–7.99%), bornyl acetate (15.80–17.33%). The main EO constitutes of flowers were Z-propenyl-sec-butyldisulfide (10.12–12.4%), eco-fenchyl acetate (10.87–12.4%), myrcisticin (13.43–15.87%) and α-bisabolol (11.23–13.76%). The primary compounds of fruit EO were β-pinene (9.32–11.26%), (Z)-1-propenyl sec butyldisulf (17.54–18.87%) and (E)-1-propenyl sec butyldisulf (28.32–30.02%). The agglomerative hierarchical clustering showed that three distinguished clusters were identified for population, where Nehbandan and Qaen had the higher similarity and Sarbisheh completely differed from the two ones. Different plant tissues contain varying types of chemicals, with leaves exhibiting higher levels of phenolic content and antioxidant capacity. This characteristic can be beneficial for industries such as food and pharmaceuticals.