Drought stress has an adverse effect on crop production and food quality. Milk thistle (Silybum marianum L.) is an oil and medicinal crop known as an alternative oil crop with high level of unsaturated fatty acids, which makes it a favourable edible oil for use in food production. Silymarin (a mixture of flavonolignans) is the main active medicinal component. Biochemical diversity, changes induced by water deficit stress in secondary metabolites, and their relationships with production traits in native germplasm are poorly understood in milk thistle. Twenty-six ecotypes mainly collected from different regions of Iran were evaluated for oil, fatty acid profile, triacylglycerol (TAG) composition, silymarin and agro-morphological traits under non-stress and water stress conditions for 2 years. Water stress increased oil and silymarin content while decreasing fruit yield and related traits. The most abundant fatty acid averaged over all ecotypes under both moisture conditions was linoleic acid (L, 39%), followed by oleic acid (O, 36%), palmitic acid (P, 9%) and stearic acid (E, 6%). Among the 24 detected TAGs, the five major compositions were OOL, OLL + OOLn (linolenic), POL, OOO, LLL + OLLn and EOL. Superior ecotypes rich in monounsaturated and polyunsaturated fatty acids were identified and can be introduced as candidates for food, medicinal and industrial purposes. Associations among different attributes are discussed.