Technical Abstract: The starch granule surface contributes the greatest proportion of surface area in a dough, and it is unknown whether starch isolated before dough development would have the same surface lipid composition as starch isolated after dough development. The compositional structure of the surface of wheat starch granules plays a crucial role in wheat endosperm hardness. The abundance of starch granule surface polar lipids is related to the physical hardness of the endosperm, but the variation in specific lipid class and molecular species is unknown. The objective of this study was to quantitatively determine the variation in polar lipids present on the surface of wheat starch granules. The experimental wheat lines used are, within each set, near-isogenic to each other but vary in grain endosperm hardness. Starch was isolated using two different processes: a dough and a batter method. Direct infusion electrospray ionization tandem mass spectrometry was used to identify and quantitatively determine the polar lipid species in wheat flour and starch. Wide ranges in starch surface polar lipid concentrations were observed between the two starch isolation methods. Starch isolation method provided a greater source of variation than wheat kernel hardness. When dough is optimally mixed, lipids originally on the surface of wheat starch become incorporated into the gluten phase of the dough, whereas in a batter system, starch surface lipids stay associated with the starch granule surface. The predominant starch surface polar lipids were di-acyl lipids of digalactosyldigylcerol (DGDG), monogalactosyldigylcerol (MGDG) and phosphatidylcholine (PC) polar lipid classes, and the predominant internal starch polar lipids were lysophosphatidylcholine (LPC) lipids