Abstract: Despite being minor components of flour, wheat (Triticum aestivum L.) lipids contribute to grain processing. They are particularly important for bread making, where they adsorb to the surface of gas bubbles formed during the proving stage of bread making, stabilizing the gas cells and improving gas retention within the dough. This contributes to the volume and texture of the loaf. However, little is understood about how their amount, composition, and properties vary in response to genotype (G), environment (E) or G × E interactions. Six wheat lines were therefore grown at three levels of nitrogen supply at Rothamsted Research, and 48 lipid species across six lipid classes were identified and quantified in white flour using electrospray ionization-tandem triple quadrupole mass spectrometry (ESI-MS/MS). This showed clear differences in the contents and compositions of lipids between cultivar as well as effects of nitrogen fertilization, which would be expected to have impacts on the processing properties of the samples.