When a corpse is found, typical questions of medico-criminal entomology involve: death time estimation or post-mortem interval determination and less frequently, the causes and place where death occurred. Entomological succession represents a powerful tool to calculate or deduce these parameters or questions. Sequences of cytochrome oxidase subunit I (COI) gene from nine species of Diptera of forensic importance registered in GenBank were used in this study. Sequences were aligned and a consensus sequence deduced for each species. A pair of oligonucleotides was designed in high conserve regions that allowed theoretical and simultaneous the co-amplification of the nine species and within this segment we used restriction enzymes (Eco57MI and BscAI) that generate different electrophoretic patterns for each species. Tentatively, with the same pair of oligonucleotides, it is possible to amplify a segment of the COI gene of nine insects of forensic importance and using a simple combination of restriction enzymes, be able to identify them. It is a technique that can be used in any laboratory with basic equipment and is an economically affordable alternative.