The New World Screwworm fly (NWS), Cochliomyia hominivorax, is an important agents of traumatic myiasis in livestock throughout Neotropical regions.  In this work the mitochondrial DNA (mtDNA) variation was used to examine the genetic variability and population structure of C.hominivorax from different geographic regions from South America. RFLP of mtDNA analysis was performed using five restriction endonucleases and there were found 20 different haplotypes for C.hominivorax. Two of the haplotypes were widely distributed in the populations, twelve were found only in a single location suggesting a reduced gene flow among some sampled populations. However, the presence of six common haplotypes among samples from Mid-west and South-east regions may suggest that there are movement of livestock with active myiasis in these Brazilian regions. PCR-RFLP was used to survey mtDNA variation in the control region (A+T-rich) of NWS from three populations from Brazil and one population from Uruguai. The control region is the major non-coding region of mtDNA and has been considered an efficient marker to characterize genetic variability within and among species. The enzyme DraI showed diagnostic patterns for the analyzed populations, although the others endonucleases tested did not showed different restriction patterns among populations. Further analysis with other enzymes that recognize sites in A+T-rich and others mtDNA regions have been conducting to elucidate the NWS intraspecific genetic variability at the geographic level.  The use of mtDNA markers has demonstrated  valuable for resolving evolutionary genetic and pest management questions that persist regarding this insect in tropical habitats. Financial support: FAPESP, CNPq.