It is unknown whether starch isolated before dough development has the same surface lipid composition as starch isolated after dough development. The abundance of starch surface polar lipids is related to the physical hardness of the endosperm, but the variation in specific lipid classes and molecular species is unknown. The objective of this study was to 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 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 on starch. Wide ranges in starch surface polar lipid concentrations were observed between the starch isolation methods. Starch isolation method provided a greater source of variation than did wheat kernel hardness. When dough is optimally mixed, lipids originally on the surface of wheat starch are dissociated, whereas in a batter system, starch surface lipids stay associated with the starch surface. The predominant starch surface polar lipids were digalactosyldigylcerol (DGDG), monogalactosyldigylcerol (MGDG) and phosphatidylcholine (PC) polar lipid classes.