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Alterations of chloroplast membrane lipids might serve as indicators of eco-physiologically induced and plant nutrition-induced changes during plant growth. The change in the degree of fatty acid saturation in the membranes is in particular a strategy of plants to adapt to abiotic stress conditions. Green multi-leaf lettuce plants (Lactuca sativa L.) were subjected to three different sulfur (S) levels. Sulfoquinovosyl diacylglycerol derivatives (SQDG) might be affected by S nutrition. Therefore, the present study was conducted to investigate the impact of S fertilization on the content and composition of individual SQDG. In addition to a change in the SQDG composition, a general change in the total lipid composition of the chloroplast membrane was observed. A significant increase in total SQDG content and doubling of the galactolipid content and significant alterations of individual SQDG were observed at elevated levels of S fertilization. High levels of S supply demonstrated a clear trend of increasing total chloroplast lipid content and concentrations of linolenic acid, in addition to a further decline in palmitic acid. The study opens perspectives on S supply and its crucial role in the build-up of photosynthetic apparatus. Moreover, it emphasizes the role of S-containing compounds, including sulfolipids, in modulating physiological adjustment mechanisms to improve tolerance ability to various abiotic stresses in plants and, consequently, plant food quality.