The Hessian fly, Mayetiola destructor (Say), poses a significant economic threat to wheat in terms of reduced grain yield, particularly in the eastern soft-winter-wheat region of the United States. However, little is know about the molecular mechanisms involved in the plant-insect interaction. The glutathione S-transferases (GSTs) form a large family of enzymes that protect cells from damage by reactive electrophilic compounds. We have cloned and characterized two GST genes from biotype GP of the Hessian fly. Sequence analysis and homology searches of the coding region for the first gene (designated mdesgst1-1) indicated it contained an intact coding region for a GST-like protein sharing homology to the insect class I GSTs. Analysis of the coding region for the second gene indicated it contained numerous stops and was most probably a pseudogene. Southern analysis and in situ hybridization on polytene chromosomes suggested the class I GSTs in M. destructor are encoded by a small gene family that is arranged sequentially on the short-arm of chromosome X1. Expression in vitro of the protein encoded by mdesgst1-1, and biochemical analysis of activity confirmed the mdesGST1-1 protein was catalytically active. Reverse transcription-polymerase chain reaction revealed mdesgst1-1 was expressed in midgut tissue, fat body, and salivary glands of larvae. Preliminary results from double-stranded RNA interference of GST gene expression seem to suggest a role for GSTs in the biochemical adaptation of M. destructor larvae to wheat.