| Summary: | Ajowan (<i>Trachyspermum ammi</i> L.) is considered a valuable spice plant with a high thymol content. Seed yield, essential oil constituents, polyphenolic composition, and antioxidant capacity of ajowan (<i>Trachyspermum ammi</i> L.) populations were evaluated in three (normal, moderate, and severe) water irrigation regimes. The highest essential oil content (5.55%) was obtained under normal condition in the Yazd population. However, both essential oil and seed yield showed significant reductions as a result of water stress. According to gas chromatography–mass spectrometry (GC–MS) analysis, thymol (61.44%), γ-terpinene (26.96%), and <i>p</i>-cymene (20.32%) were identified as the major components of the oil. The highest (89.01%) and the lowest (37.54%) thymol contents were in Farsmar and Hamadan populations in severe stress condition, respectively. Based on HPLC analysis, chlorogenic (3.75–47.35 mg/100 g), caffeic (13.2–40.10 mg/100 g), and ferulic acid (11.25–40.10 mg/100 g) were identified as the major phenolic acids, while rutin was determined as the major flavonoid (11.741–20.123 mg/100 g). Moreover, total phenolic and flavonoid contents were elevated under drought stress treatment, while antioxidants responded inconsistently to stress based on two model systems. Overall, the Yazd population exhibited a superior response to water stress, as evidenced by its less reduced thymol and oil yield content, while Arak and Khormo had the highest accumulation of polyphenolic compounds.
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