Ninety-four microsatellites from enriched genomic libraries of Hessian fly (Hf, Mayetiola destructor [Say]) were localized to 170 cognate clones in an Hf bacterial artificial chromosome (BAC) library. These microsatellite-positive BAC clones were physically mapped to polytene chromosomes by fluorescent in situ hybridization. The mapped microsatellite loci can be used to study the genetic diversity and population structure of Hf.