Long-PCR strategies have significantly improved the sequencing and characterization of insect mitochondrial genomes essentialy by providing an alternative way to purify sufficient mtDNA from limited amounts of individual template and thus stimulating insect molecular and evolutionary biology research. We optimized Long-PCR reactions for the amplification and sequencing of the complete mitogenome of the horn-fly, Haematobia irritans (Diptera: Muscidae), a key parasitic fly of cattle. H. irritans complete mitogenome was amplified using four conserved Long-PCR primers designed on Dipteran 16S lrDNA gene (H16S series) combined with two previously described ‘universal’ primers (Simon et al. 1994). Amplified products ranged from 8.5 to 9.3Kb depending on primer combination, with a 1.5Kb overlap. We obtained specific amplicons on a microgram range providing sufficient mtDNA for several molecular biology techniques, including RFLP analysis, mtDNA probe construction and shotgun sequencing of mitochondrial genomes. On-going sequencing of the Horn-fly mitochondrial genome using this approach provides an informative background that optimizes the characterization of complete mitochondrial genomes of other Dipteran species. Using H16S primers, we also efficiently amplified long-PCR products from eleven other Calyptratae key species of medical-veterinary importance: the screw-worm fly, Cochlyomyia hominivorax, the blowflies Cochlyomyia macellaria, Chrysomya albiceps, Chrysomya megacephala, Chrysomya putoria, Lucilia cuprina, Phaenicia eximia, the botfly, Dermatobia hominis and the muscids, the house-fly, Musca domestica, the stable-fly, Stomoxys calcitrans and Ohpyra aenescens. These results may contribute not only to achieve complete Haematobia irritans mitochondrial genome sequencing and characterization but also stimulate other Calyptratae mitochondrial genomics. Financial support: CNPq/PROFIX and FAPESP