The phylogeographical history of the pest fly screwworm, Cochliomyia hominivorax (Coquerel), was studied using partial mitochondrial DNA sequences of the control region, Cytochrome c oxidase (CO) subunit I and CO subunit II from 361 individuals collected across its current geographic range. Analyses showed marked genetic differentiation on a macrogeographic scale. The genetic diversity in the species is structured into four main “regional groups,” corresponding to Cuba, the Dominican Republic, and the North and South Amazon region. Results indicated that the distribution of screwworm genetic diversity was mainly shaped by historical events, i.e., colonization of Caribbean islands, vicariance in the Amazon region and population expansion. Demographic history analyses revealed that the population expansion started ≈20–25,000 yr ago and recently increased exponentially. We hypothesized that the initial period of expansion was probably associated with environmental amelioration in the late Pleistocene and the exponential increase with resource availability in recent times. The population expansion is probably responsible for the low divergence and the lack of genetic and geographic correlation in the South Amazon region but did not erase the genetic structure pattern on a continental scale. The screwworm is one of the most damaging livestock pests in South and Central America, and the pattern of genetic variability distribution reported here suggests that the Caribbean area and the North and South Amazon regions could be considered as independent units for future pest control programs.