Specific regions of the mitochondrial DNA (mtDNA) have been used as efficient molecular markers in many evolutionary studies concerning a wide diversity of animal taxa, providing informative data for understanding both organismal and molecular evolution. This work reports the molecular characterization of the cytochrome oxidase subunit I (COI) gene and the control region (CR or A+T-rich region, in insects) of the mtDNA of the species Haematobia irritans (horn fly), Stomoxys calcitrans (stable fly), and Musca domestica (house fly) (Diptera: Muscidae). Universal insect mtDNA primers were used to recover both regions of these species, except for the S.calcitrans CR, which required specific strategies for PCR optimization. The A+T composition of the COI gene (1473bp) in these Muscidae species was the highest described among Diptera (from 69,4% to 71,3%). Comparative analysis of the predicted COI aminoacid sequences resulted in substitution patterns in agreement with the evolutionary model previously described for the insect COI proteins. The analysis of H.irritans (1256bp) and M.domestica (1287bp) CRs showed high levels of nucleotide sequence variation. The conserved sequence blocks previously described in myiasis-causing flies (Calliphoridae and Oestridae) were recognized in H.irritans and M.domestica CR sequences, despite the hypervariable nature of this region, suggesting that Muscoidea and Oestroidea mtDNA might share a similar replication/transcription regulatory system. In addition to the interest in the analysis of the patterns of molecular evolution of mitochondrial genome, the characterization of specific mtDNA regions may contribute in further investigations of intra and interspecific variability of these important pest species. Financial support: PROFIX/CNPq, FAPESP.