The family Calliphoridae is formed by myiasis-causing flies, being known for its economic, medical, sanitary and forensic importance. Ecological similarities and the lack of morphological diagnostic traits may cause problems in the identification of species, especially for larvae. The mitochondrial DNA (mtDNA) is provinding important clues for species identification and variability characterization. The control region (CR) represents the major noncoding region of the mitochondrial genome, with a bias towards A+T content. The CR has been divided in two different subregions, including the A domain with conserved sequence blocks and a hipervariable B domain. The entire CR could be recovered in two separated PCR reactions using a specific and universal primers combination. The efficient PCR amplification, sequencing and analysis of nine Calliphoridae species has shown a complete tRNA ^Ile duplication into the B domain of five Chrysomya species: C. albiceps, C. bezziana, C. chloropyga, C. megacephala and C. rufifacies. The tRNA^Ile duplication could be used as a diagnostic marker for Chrysomya genus identification, since the duplication has not been reported for other related Calliphoridae genus. The characterization of the A domain for Calliphoridae species will allow the evaluation of this region as a molecular marker for evolutionary studies and for identification of Calliphoridae species.