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Ajowan (Trachyspermum ammi) is an industrial medicinal plant with different food and pharmaceutical applications. Essential oil yield and composition of 24 ajowan populations in different locations of Iran was investigated. The essential oil content of populations ranged from 1.68% in Yazshah to 5.55% in Yazd. According to GC-MS analysis, thymol (48.84-61.44%), gamma-terpinene (26.96-19.46%) and p-cymene (15.09-20.32%) were the major components. According to hierarchical cluster and principal component analyses, two chemotypes were determined for Iranian ajowan populations, including high thymol, and high gamma-terpinene and p-cymene groups. The highest correlation coefficient was between beta-pinene and alpha-pinene (+0.709), while the highest negative correlation was between gamma-terpinene and thymol (-0.896). In the present study, the climatic and soil orders of studied populations were determined. Canonical correspondence analysis (CCA) was also applied to assess the impact of geographic variation and environmental conditions on chemical variability of ajowan. Finally, it can be suggested that high essential oil and thymol content (phenolic chemotype) can be obtained in poor, undeveloped, relatively stony and shallow soil orders in arid and semi-arid conditions, while none phenolic ones needs higher humidity and soil depth that the first group. (C) 2016 Elsevier B.V. All rights reserved.