Full-length cDNA and genomic sequences for two genes (designated mdesprot-I and mdesprot-II) encoding digestive serine proteases in Hessian fly, Mayetiola destructor, have been cloned and characterized. The deduced amino acid sequences revealed similarity with trypsin-like digestive serine proteases from other Dipterans. Both mdesprot-I and mdesprot-II encoded proteins with secretion signal peptides at the N-terminals, indicating the proteins are secreted proteases that should function as midgut digestive proteases. A cytological analysis with fluorescent in situ hybridization revealed the cytological localization of mdesprot-I and mdesprot-II on the long arm of Autosome 2. Results are discussed in the context of the efficacy of potential protease inhibitors to develop Hessian fly resistant wheat through genetic engineering approaches.