In the present research, 28 populations of ajowan (Trachyspermum ammiL.) were evaluated for agro-morphological traits and essential oil yield in two consecutive years. Then, selected ajowan populations from these two years were used for further morphophysiological and biochemical studies under different salinity levels (control, 60, 90, and 120 mM NaCl). The main components of the oil were thymol (32.7–54.29%), γ-terpinene (21.71–32.81%), andp-cymene (18.74–26.16%). Salt stress caused an increase in essential oil content in the Esfahfo and Qazvin populations. The highest total phenolic and flavonoid contents were found in the Arak population grown in 60 mM NaCl (183.83 mg TAE g−1DW) and the Yazd population grown in 90 mM NaCl (5.94 mg QE g−1DW). Moreover, the Yazd population exhibited the strongest antioxidant activity based on DPPH (IC50= 1566 µg/mL) under 60 mM NaCl and the highest reducing power (0.69 nm) under 120 mM NaCl. The results revealed that low and moderate salt stress improves the phytochemicals of ajowan seeds, which are useful for pharmaceutical and food applications. In this research, some morphological traits, as well as essential oil yield, were evaluated in open pollinated versus self-pollinated plants. As a result, plant height, number of flowering branches, and crown diameter significantly decreased in some populations, while a significant increase was obtained for number of flowers per umbel and seed numbers per umbel. Finally, self-pollination of ajowan might provide new insights for further breeding programs to increase oil or thymol content in ajowan.