Domain III of the mitochondrial 12S rRNA gene from Hessian fly, Mayetiola destructor (Say), was sequenced in 21 populations from the United States, two populations from Canada, five populations from the Mediterranean basin, one population from Southwest Asia, and one population from New Zealand. From the total alignment, seven unique mitochondrial DNA (mtDNA) sequences (haplotypes) were identified. Of the seven 12S haplotypes only three (haplotypes 1, 2, and 3) occurred in populations from North America, indicating these were most likely the introduced haplotypes. Haplotypes were not restricted to any given biotype of M. destructor as defined by virulence to resistance genes in wheat. Thus, neutral markers did not show structure based on biotype. Populations of M. destructor showed a predominance of one haplotype over the others in specific geographic regions. However, Wolbachia DNA was not detected in any population, indicating that inheritance patterns of mtDNA in M. destructor were most likely due to repeated bottlenecks leading to the expansion of one lineage over another. The complete complement of 12S sequences in the M. destructor populations was subjected to a phylogenetic reconstruction by using haplotypes 1 and 3 of the gall midge Orseolia oryzae (Wood-Mason), as outgroups. Results from this initial study indicate a more robust phylogenetic reconstruction and analysis of population history will test the hypothesis of a single introduction of M. destructor into North America.