The blowflies of the Calliphoridae family (Diptera: Brachycera) are a group of medical, veterinary and forensic importance, known as myiasis-causing flies. The carrion-breeding species of this family are vectors of pathogens because they feed and breed in feces, garbage and corpses. Molecular markers are currently used in Calliphoridae evolutionary studies, mainly applied to forensic entomology, population genetics and phylogenetic inferences. In support of this scenario, the internal transcribed spacer 2 (ITS2) is been characterized for Calliphoridae species. The ITS2 comprises a small sequence (<400bp) located inside the nuclear ribossomal DNA (rDNA) cluster, flanked by the 5.8S and 28S rDNA subunits. This region has a high-copy number in the nuclear genome, presenting a suitable number of variable characters that may be useful for phylogenetic analysis and species identification. The presence of a secondary structure, required for the correct processing and folding of the functional rRNA subunits, provides additional characters that already had been used for phylogenetic reconstructions in closely related taxa. The ITS2 of twelve Calliphoridae species have been sequenced after amplification by PCR and cloning. The sequences obtained showed the presence of an additional rRNA 2S subunit, located inside the ITS2, feature firstly described for Drosophila melanogaster. The rRNA 2S splits the ITS2 into ITS2a (~30bp) and ITS2, ranging in size from 296bp to 350bp for the species analized. The secondary structure analysis is being conducted and will provide more information about conserved and variable regions inside the ITS2, contributing for its evaluation as a reliable molecular marker within Calliphoridae.