The advance of large-scale sequencing technologies and the power of newly developed databases are drastically improving the current state of arthropodan mitochondrial genomics. Mitochondrial genomes are a powerful dataset for comparative studies. Brazil has played a major role on the structural characterization of mitochondrial genomes from species of great medical and veterinary importance (such as the blowflies, Cochliomyia hominivorax, Chrysomya chloropyga, Dermatobia hominis and, more recently, the parasitic Muscidae flies Haematobia irritans and Stomoxys calcitrans). This work aims to add a contribution from the analysis of the horn fly mitochondrial genome (GenBank NC_007102). Methodological strategies involved 2-steps Long-PCR reactions, shotgun library, sequencing and assembling the genome using phred/phrap/consed and global comparative analysis on AMiGA (Arthropodan Mitochondrial Genomes Accessible database – http://amiga.cbmeg.unicamp.br). The horn fly genome has 16.079 pb in size and is 79.1% A+T-rich, becoming the third A+T-richest mitochondrial genome of dipteran species. Its gene content and organization are conserved regarding the ancestral model proposed for Diptera. The control region (1259 bp) showed conserved motifs homologous to those previously described in Calyptratae, suggesting a common regulatory system. The presence of a coding-probability for a second trnF in the opposite strand of a trnE confirms the plasticity of these genetic elements in the animal mitochondrial genome. This is the first Muscidae mitochondrial genome described to date, which contribution includes providing an informative source of data for comparative studies, designing ‘taxon-specific’ primers, mapping restriction sites and optimizing PCR-RFLP analysis and encouraging studies on molecular evolution and mitochondrial genome diversity. Financial support: CNPq/Profix, FAPESP.