Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure of screwworm flies in four populations from São Paulo State, Brazil. The total DNA of 405 individuals was digested with 15 restriction endonucleases and probed with five cloned HindIII fragments representing the entire mitochondrial genome of Cochliomyia hominivorax. The survey revealed that four enzymes (HaeIII, HindIII, MspI, and PvuII) were suitable to detect mtDNA variation among all populations. Based on the fragment patterns obtained for these four enzymes, a total of 15 haplotypes in combination was detected. Heteroplasmic individuals for the PvuII pattern were obtained in one of the populations. The estimated average for nucleotide sequence divergence (delta) was 0.92%. The cladogram of the geographical distribution among the observed haplotypes suggests that the sampled screwworms probably belong to a single evolutionary lineage with populations interconnected by reduced gene flow.33237-56