The New Word screwworm, Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is one of the most important insect pests of livestock in the Neotropical region. In this work, polymerase chain reaction-restriction fragment length polymorphism of mitochondrial DNA (mtDNA) was used to study the diversity and population structure of seven geographically distinct populations of C. hominivorax from most of the important livestock areas in Uruguay. The control region (A T/12S) and subunits 1 and 2 of cytochrome oxidase (cox1/cox2) were amplified and digested with restriction endonucleases. Nine haplotypes were observed among the populations sampled. The mean nucleotide diversity and the haplotype diversity indicated high mtDNA variability in this species. The similarity index, average nucleotide divergence, and analysis of molecular variance results showed no evidence of subpopulation differentiation, indicating that the C. hominivorax populations of Uruguay form a single panmitic population. The distribution pattern of the genetic variation in natural populations of C. hominivorax and the implications of these results for establishing control program are discussed.