Analysis of structure and evolution of mitochondrial DNA (mtDNA) regions has contributed for the identification of molecular markers for evolutionary studies. This work reports the characterization of two mtDNA regions in the horn fly, Haematobia irritans, the COI gene and the control region (CR). These regions were successfully amplified using insect mtDNA universal primers resulting in 814bp (COI) and 2000bp (CR) amplicons. Both regions were sequenced after cloning and by direct sequencing of PCR products, providing confident results for any of the applied strategies. Analyses of H.irritans CR sequences identified flanking regions and the main conserved motifs previously described for other dipterans. The CR has nearly 1200bp and lies between rRNA12S and tRNA^Ile genes. A 670bp region of the 12S gene and 150bp of the tRNA gene cluster (IQM) were also sequenced and analyzed. Significant bias towards A«T transversions agrees with the model of insect mitochondrial genome evolution. Conserved elements associated to the CSB I, III and IV previously described in myiasis-causing flies were identified in H.irritans. These elements may be related to the regulation of replication and transcription processes of the mtDNA. Sequence alignment at hypervariable domains provides no confident results to validate homologous sites among Calyptratae species. Carboxy-terminal region of COI was sequenced and compared with other Muscoidea species. Five aminoacids replacements were identified, which agree with the model of structural evolution suggested for insect COI protein. This study provides informative data for further population, pest-management and evolutionary investigations for this important parasitic fly. Financial support: CNPq/PROFIX, FAPESP.