The New World Screwworm fly, Cochliomyia hominivorax (Diptera: Calliphoridae), is an important agent of traumatic myiasis in livestock throughout Neotropical regions, causing great profit looses to cattle breeders. In this work, PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) of mitochondrial DNA (mtDNA) was used to examine diversity and population structure of thirteen geographically distinct populations of C. hominivorax from Brazil and Uruguay. The control region (A+T/12S) and subunits 1 and 2 of cytochrome oxidase (cox1/cox2) were amplified and digested with restriction endonucleases. Ten haplotypes were observed among the populations sampled, three haplotypes were widely distributed and the remaining locally distributed. The mean nucleotide diversity and the haplotype diversity indicated high mtDNA variability in this species. The AMOVA results showed low subdivision in Central Brazil populations and no evidence of subpopulation differentiation in subtropical region. Further analysis with other populations from Brazil and other South American countries is being conducted to elucidate the C. hominivorax intraspecific genetic variability at the geographic level. 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. Financial support: FAPESP, CNPq, IAEA.