Background and aim: Mechanisms of drought tolerance based on root architecture and lipid composition in wheat are poorly understood. We quantified the differences in root morphological traits and phospholipids and galactolipids levels between winter and spring wheat genotypes at variable water supply amounts (drought stress). Methods: Experiments were conducted using seven winter and four spring wheat (Triticum aestivum) genotypes. In the first experiment, solid agar medium was used to quantify seminal root angles. In the second experiment, the plants were grown in 150-cm columns in a greenhouse under full and deficit water supply for 65 days to record root architecture. The root tips (2-cm-long) were used for quantifying polar lipids. Results: Drought stress at vegetative stage decreased plant height (14%), total dry matter production (48%), maximum root length (25%), root length:shoot length ratio (11%), and other root traits. Winter wheat genotypes had ~1.5 times higher maximum root length than spring wheat genotypes. Significant differences in molar percentages of root phospholipids and galactolipids, molecular species, and double bond index of galactolipids were observed among spring wheat but not winter wheat genotypes. Conclusions: Based on the genotypes studied, the drought tolerant mechanism of winter wheat was associated with deep root system, and in spring wheat it was well branched (albeit shallow) root system with more unsaturated membrane lipids.