Hessian fly, Mayetiola destructor (Say), is a wheat crop pest in the United States creating millions of dollars of crop losses each year. The primary means of controlling this insect pest is through the use of resistant cultivars. Over the years, this practice has led to selection pressures that have allowed for virulence genes to evolve. Additionally, other characteristics about Mayetiola destructor biology are likely to reduce gene flow between populations, such as limited dispersal, short life span, and rapid mating and oviposition; however, virulence genes continue to emerge and spread. Knowledge of genetic variation within and between populations is important for monitoring the effects of any insect targeted control strategy, including the spread of virulence to deployed resistance genes in wheat, yet interactions on the population level for this insect remains poorly understood. In order to better understand Hessian fly on the population level, microsatellite markers were used to identify the population structure of 10 Hessian fly collections from the Southeastern United States. Levels of variation within and between populations are examined and the possible implications for the Hessian fly populations in this region are explored.