Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this work, we analysed the molecular species composition of phosphatidylglycerol (PG) in thylakoid membranes during spinach (Spinacia oleracea) and squash (Cucurbita pepo) cotyledon growth to investigate how the growth temperature affects the PG biosynthesis. Of the 10 molecular species detected, temperature affected mainly the relative content of molecular species containing linolenic acid (18:3) and those containing palmitic acid (16:0) at the sn-1 position of glycerol backbone. Lowering the temperature induced an increase in the former and a decrease in the latter. The relative content of molecular species containing 18:3 or 16:0 at the sn-1 position of the glycerol backbone were correlated with temperature. Our results indicate that the substrate selectivity of the glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts towards 16:0 or oleic acid (18:1) and the activity of fatty acid desaturases are greatly affected by temperature. In addition, changes in the relative content of PG molecular species induced by variations in growth temperature depended mainly on the substrate selectivity of GPAT.