Background <p>In this study, we investigated the genetic variability and population structure of the New World screwworm fly <i>Cochliomyia hominivorax</i>. We tested the hypothesis that the species exhibits a center–periphery distribution of genetic variability, with higher genetic diversity in central populations (e.g., Brazil) and lower diversity in peripheral populations.</p> Methods <p>Using microsatellite markers, we analyzed larvae collected from infested livestock across South America. The larvae were collected directly from various wound sites to ensure a broad representation of genetic diversity.</p> Results <p>Contrary to our initial hypothesis, the results revealed consistent genetic variability across the species’ distribution, low population differentiation, and no evidence of isolation-by-distance patterns among subpopulations. The genetic analysis indicated an excess of homozygotes, potentially due to the Wahlund effect, null alleles, or selection pressure.</p> Conclusions <p>These findings suggest a complex metapopulation structure for <i>C. hominivorax</i>, challenging classical population genetics models. This complexity likely arises from the species’ high dispersal capability and frequent local extinctions followed by recolonization. These results have important implications for the design and implementation of control programs, emphasizing the need for coordinated and large-scale actions rather than isolated initiatives.</p> Graphical Abstract <p></p>