Mayetiola destructor (Say) (Diptera: Cecidomyiidae), the Hessian fly, is distributed across most of the wheat (Triticum aestivum L.)-growing areas of the world and can cause significant yield losses in wheat production. Native to the Old World, Hessian fly reportedly came to the United States in a single introduction during the Revolutionary War and has since spread across North America. Using a nuclear marker, the population structure of North American Hessian fly was examined with regard to collections from the Old World. White intron 1 (wint1) showed insignificant recombination within collections and a large number of informative characters, allowing its use as a phylogenetic marker. Thirty-three alleles of wint1 were identified. Population structure analyses divided the collections into four populations: Middle East, Old World I, Old World II, and New World. More variation was found within populations than between populations, indicating that gene flow exists between local areas. However, shared ancestral characteristics resulted in mixing of Hessian fly collections into more than one population as revealed by the population structure reconstruction. North American and Spanish collections were a mixture of the Old and New World populations. With the sharing of ancestral characters as well as wint1 alleles, this study indicates that the North American lineage may actually be associated to an unsampled location, perhaps northern Europe. If the single introduction hypothesis were correct, then both lineages would have to have been present at the time of introduction.