The biosynthesis and accumulation of secondary metabolites in plant tissues interact strongly with environmental conditions and breeding systems. Limited knowledge is available on the effects of breeding system (self vs. open pollination) and deficit irrigation on the composition of flavonolignans, seed yield, and oil content of different genotypes of milk thistle (Silybum marianum L.). Four ecotypes of Iranian milk thistle collected from diverse geographical regions were each self-and open-pollinated; they were then assessed in the field under both normal and deficit irrigation for seed yield, oil percentage, silymarin, and its components during 2014 and 2015. Deficit irrigation decreased seed yield and silybin A but increased total silymarin content, silybin B, and silydianin. In both moisture environments, seed yield was positively associated with oil percentage and silybin A. Open pollination improved seed yield, oil percentage, total silymarin, and its components in milk thistle ecotypes as compared with self-pollination. Considerable genetic variability was observed among the evaluated ecotypes in their response to moisture environments and pollination status. Under open pollination, the northern Iranian ecotypes Mashhad and Sari were identified as promising varieties for further studies.