Milk thistle (Silybum marianum L.) is an annual medicinal plant whose seeds contain silymarin, a valuable bioactive compound widely used in the treatment of liver disorders and other diseases. Because Iran is characterized by extensive saline soils and limited freshwater resources, identifying salt-tolerant milk thistle populations is essential for sustainable cultivation in salt-affected areas. This study evaluated the salinity tolerance of six milk thistle populations under different irrigation water salinity levels during the 2024–2025 growing season at the Sadouq Research Farm of the National Salinity Research Center. The experiment was conducted as a split-plot based on a randomized complete block design with three replications. Four irrigation water salinity levels (3, 6, 9, and 12 dS/m) were assigned to the main plots, while six populations (Golestan, Isfahan, Fars, Mazandaran, Ardabil, and Khuzestan) were allocated to the subplots. Measured traits included plant height, thousand-seed weight, inflorescence yield and diameter, number of seeds per inflorescence, number of inflorescences per plant, seed yield, shoot dry weight, and harvest index. Salinity significantly affected all measured traits, whereas population effects were significant for most traits. The salinity × population interaction was significant only for plant height. Plant height decreased from approximately 70 cm in the Ardabil, Fars, and Golestan populations at 3 dS/m to about 30 cm in the Fars and Mazandaran populations at 12 dS/m. Severe salinity reduced thousand-seed weight by 50%, while inflorescence yield, inflorescence diameter, seed number per inflorescence, number of inflorescences per plant, and seed yield decreased by 40%, 22%, 33%, 48%, and 53%, respectively. Overall, the Ardabil, Fars, Golestan, and Mazandaran populations exhibited greater salinity tolerance than the Isfahan and Khuzestan populations, highlighting their potential for cultivation and breeding programs in saline environments.