Species identification of the early colonizers on a corpse, such as Calliphoridae and Sarcophagidae, provides useful information for estimating the postmortem interval (PMI). However, the morphology of adult flies and nymphs is very similar among species of the same genus, and for many forensic scientists accurate identification is very difficult within their routine work. We present a simple and time-saving method for identification of forensically important flies by comparing sequenced 304 bp of mitochondrial DNA cytochrome oxidase gene subunit I (mtDNA COI). Eggs and larvae were recovered from the corpse during forensic autopsies and then reared in a growth chamber under controlled conditions. The emerging adults were killed by ethyl acetate and male specimens were observed under a stereomicroscope to identify the species based on the shape of the genitalia. Subsequently, DNA was extracted from identified specimens and sequenced mtDNA COI, resulting in the identification of 8 species of 4 genera of forensically important diptera. Moreover, the molecular biological species identification was useful in forensic practice, and there were cases in which analysis of DNA extracted from puparia supposed to be early colonizers of the corpse provided useful information for estimating the PMI. We also report sequencing of mtDNA COI of the green bottle fly, Lucilia ampullacea, newly collected from a corpse in Iwate prefecture.