The New World screwworm fly Cochliomyia hominivorax (Coquerel, 1858) (Diptera: Calliphoridae) is an important livestock pest endemic to the Americas that has been eradicated from North and continental Central America with a control program based on the Sterile Insect Technique (SIT). The establishment of target management units is a strategic step in the implementation of new control programs, which can be achieved using genetic studies of natural populations. Previous studies of New World screwworm fly populations were conducted on the continental scale and identified four main groups: two in South America and two in the Caribbean. However, studies within these groups are needed to determine which smaller geographic areas can be treated as management units. Here, we analyze the genetic variability distribution and the population demographic signals of the New World screwworm fly in a 6,000 km² area located along the border of Brazil and Uruguay. This area was the subject of the first control pilot program conducted in South America. We studied eight microsatellite loci and sequences from two mitochondrial DNA regions in individuals sampled at 20–25 livestock breeding farms. We observed no population structure and found high genetic variability on the geographical scale sampled for both molecular markers. Our microsatellite data suggest that these populations are not in equilibrium, and demographic analyses based on mitochondrial data indicate population expansion.These results suggest that this geographic scale is not adequate for future New World screwworm fly management in South America.