Technical Abstract: Hessian fly (Mayetiola destructor) is a biotrophic parasitic insect that interacts with wheat on a typical gene-for-gene basis. In this study, we systematically profiled changes in membrane lipids in two isogenic wheat lines: a susceptible line and its backcrossed offspring containing resistance gene H13. Our results revealed a 32 to 45% reduction in total concentration of 129 detected lipid species in resistant plants during incompatible interactions within the first 24 hrs after the initial attack. No significant change was observed in lipid concentration in susceptible plants during compatible interactions. Microarray and quantitative PCR analyses of 168 lipid-metabolism related transcripts, including those encoding various lipases, lipid transfer proteins, enzymes involved in oxylipin synthesis, and enzymes involved in wax and cutin synthesis, revealed that the levels of many of these transcripts increased rapidly in resistant plants after Hessian fly attack, but did not change in susceptible plants. Collectively, our data, along with previously published results, indicate that the mobilized lipids were converted through lipolysis into fatty acids, which were further converted into defense-signaling molecules including oxylipins and direct-defense molecules such as components of cuticle wax. Our results suggest that rapid mobilization of membrane lipids constitutes an important step in wheat defense against Hessian fly.