The greenhead horse fly, Tabanus nigrovittatus Macquart (Diptera: Tabanidae), is frequently found in coastal marshes of the Eastern United States. The females are autogenous (i.e., able to develop eggs without a bloodmeal), but they become a considerable pest to both humans and animals when they pursue a source of blood protein to produce additional eggs. In this study, we identified microsatellite markers to provide first insight into the population genetic structure of this notorious pest species. Because no prior genomic information was available for T. nigrovittatus,weused direct shotgun pyrosequencing technology to characterize microsatellite loci. Approximately 10% of the 105,634 short sequence reads generated from random genome sampling contained microsatellites with at least four repeats of di-, tri-, tetra-, penta-, and hexamers. Primers were designed for 36 different microsatellite loci with di-, tri-, and tetramer repeat units. After optimization, 20 primer pairs yielded consistent PCR products and were validated for population genetic application in six populations in Western Louisiana. Ten loci were polymorphic with 2–9 alleles per locus and an average observed heterozygosity of 0.20 across populations. The horse fly populations from different trap sites (distance 50–144 km) or years of collection (2010 vs 2011) were genetically distinct from each other (F_ST = 0.05–0.39) and genetically diverse (gene diversity: 0.24–0.37) but considerably inbred (FIS: 0.22– 0.47), with high mean relatedness among individuals (r = 0.27), suggesting the capture of a high percentage of sisters at the same trap location who were progeny of incest.