Transient heat stress compromises resistance of host plants to Hessian fly, Mayetiola destructor (Say), and other biotic stresses. However, the mechanism for the loss of plant resistance under heat stress remains to be determined. In this study, we determined polar lipid profiles in control and Hessian fly-infested resistant and susceptible wheat seedlings with and without heat stress using an automated electrospray ionization tandem mass spectrometry analysis. Heat stress, alone or in combination with Hessian fly infestation, caused significant reduction in the abundance of total detected polar lipids and double bond index. Changes in lipid profiles in ‘Molly’ were similar to those in ‘Newton’ under heat stress. However, changes in lipid profiles in Molly were significantly different from those in Newton following Hessian fly infestation. The combination of heat stress and Hessian fly infestation resulted in unique lipid profiles in comparison with those in plants either treated with heat stress or infested with Hessian fly alone. In addition, a greater impact on lipid metabolism was observed in heat-stressed plants infested with Hessian fly than that in plants treated with either heat stress or Hessian fly alone. Our results suggest that changes in lipid metabolism caused by heat stress may be part of the metabolic pathways through which heat stress suppresses resistance of wheat plants to Hessian fly infestation.