The bot fly Dermatobia hominis is a parasite of humans and livestock, producing a furuncular myiasis. The larvae of D. hominis develops in the subcutaneous tissue causing economic loss and health problems, being one of the most important livestock pests in Latin America. The complete mitochondrial DNA (mtDNA) sequence of D. hominis was determined and has 16360 bp. The nucleotide composition is biased towards adenine and thymine, which accounted for 77.8% of the whole genome. The cox1 gene has a Serine as the start codon, while incomplete termination codons were found for cox1, cox2 and nd5. The control region, protein-coding genes, rRNA and tRNA genes are in the same order and orientation when compared to other dipteran mitochondrial genomes, except for a duplication of tRNA^Val located approximately in the same position of the tRNA^Asp, but in the opposite strand. The identification of restriction sites on the sequenced mtDNA and the correlation with previous RFLP analyses provided insights for an overall scenario of the genetic variability and population structure of D. hominis, being helpful for monitoring the variation and dynamics of this species. The availability of complete mitochondrial sequences is an important source of sequence information for Diptera molecular and evolutionary approaches.