A high performance liquid chromatographic (HPLC) method was developed for analyzing digalactosyldiglycerides (DGDG) and monogalactosyldiglyceride (MGDG) in polar lipids fractionated from lipid extracts of wheat or flour. Wheat lipid samples were prepared by solvent extraction, then fractionated on a silica gel packed open column. A Spherisorb ODS (octadecyl silane) column with methanol/water elution system was used for separation of glycolipids in the polar lipid fractions. The detection limit of the refractive index detector with interferometric optics was 0.25µg for both DGDG and MGDG. Separating on nonpolar bonded phase columns permitted us to differentiate, based on fatty acid composition and position, among components within the specific glycolipid classes. Semipreparative HPLC on analytical columns was used to subfractionate the polar lipids. The glycolipids were collected for functional group characterization. Approximately 35% of each DGDG subfraction was accounted for as carbohydrate. The absence of phosphorus precluded phospholipids. Fatty acid analysis by gas chromatography showed the first DGDG to be linoleic acid, whereas the second DGDG peak was composed of linoleic, oleic and palmitic acids. Mass spectrometric analysis of the first DGDG peak showed linoleic acid in both the SN-1 and 2 positions. Mass spectrometric analysis revealed that palmitic or oleic acid in the second peak was preferentially located on the SN-1 position; linoleic acid was on the SN-2 position