Technical Abstract: Reactive oxygen species (ROS) play a major role in plant defense against pathogens, but evidence for their role in defense against insects is still preliminary and inconsistent. In this study, we examined the potential role of ROS in defense of wheat and rice against Hessian fly (Mayetiola destructor) larvae, model systems to study gall midge – plant interactions. Rapid and prolonged accumulation of H2O2 was detected in wheat plants at the attack site during incompatible interactions. Increased accumulation of both H2O2 and superoxide was detected in rice plants during non-host interactions with the larvae. No increase in accumulation of either H2O2 or superoxide was observed in wheat plants during compatible interactions. The low concentration (LD50<0.05 µg/ml or 1.7 µM) of H2O2 required for lethality to Drosophila larvae suggested that the ROS accumulation at the attack site could cause toxicity to Hessian fly larvae. A global analysis revealed changes in the abundances of 243 wheat transcripts and 313 rice transcripts encoding proteins potentially involved in ROS homeostasis. A large number of transcripts encoded class III peroxidases that increased in abundance during both incompatible and non-host interactions, whereas the levels of these transcripts decreased in susceptible wheat during compatible interactions. The higher levels of class III peroxidase transcripts was associated with elevated enzymatic activity of peroxidases at the attack site in plants during incompatible and non-host interactions. These data indicated that class III peroxidases may play a major role in ROS generation in resistant wheat and non-host rice plants during response to Hessian fly attacks.