In higher plants, glycolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are major components of chloroplast membranes in leaves. A recent study identified an isoform of MGDG synthase that is expressed specifically in floral organs, suggesting a novel function for glycolipids in flowers. To elucidate the localization and developmental changes of glycolipids and their biosynthetic activities in flowers, we carried out a series of analytical studies with Petunia hybrida. The results showed that the biosynthetic activities of galactolipid synthesis, particularly for DGDG, increased during flower development. Among the floral organs, the pistil had the highest galactolipid synthetic activity. Its specific activity for incorporation of UDP-galactose to yield galactolipids was estimated to be more than twice that of leaves, which are the major site of galactolipid synthesis in plant tissues. Analysis of lipid contents of pistils revealed that they contained higher amounts of galactolipids than other floral organs. Moreover, DGDG was more abundant than MGDG in both pistils and petals. These results show that DGDG is a major glycolipid in floral organs and that DGDG biosynthetic activity is highly upregulated in the pistils and petals of Petunia flowers.