The primary screwworm fly Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important insect pests of livestock in neotropical regions. In this work, PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) of mitochondrial DNA (mtDNA) was used to study the diversity and population structure of seven geographic populations from Uruguay. The Control Region and the subunits I and II of Cytochrome Oxidase were amplified and digested with diagnostics enzymes. Nine haplotypes were observed among the C. hominivorax samples examined. Mean nucleotide diversity (π=2,3%) and haplotype diversity (H=0,6533) indicated high mtDNA variability in this specie. The similarity index (96,7%), the nucleotide divergence (d=0,00055) and the AMOVA result showed no evidence of subpopulation differentiation, indicating that the populations of Uruguay are a unique panmitic population. The characterization of NWS intra specific genetic variability at the geographic level is necessary to monitoring the expansion in the current distribution and into screw-worm free areas. Financial support: FAPESP, CNPq, IAEA.