Many species of Muscidae are cosmopolitan synanthropic insects. It is frequently found on dead bodies after death, so an accurate identification of each species is necessary for the correct estimation of the postmortem interval (PMI). Identification species of Muscidae is traditionally performed through their morphological features. However, the morphological similarity of Muscidae in the level of species usually poses a challenge for forensic entomologists. Therefore a molecular method used 16S rRNA sequence on mitochondrial DNA was established for species identification. In this study, a 289 base pair region of mitochondrial DNA (mtDNA) coding for 16S rRNA was investigated for identification of the following forensically important species of Muscidae. The specimens were from four genera, including 18 Musca domestica (Linnaeus, 1758), 2 Ophyra spinigera (Stein, 1910), 2 Ophyra chalcogaster (Wiedemann, 1824), 1 Morellia hortorum (Fallén, 1817), and 1 Fannia canicularis (Linnaeus, 1761). 16S rDNA region allowed the identification of these species of Muscidae, indicating the possibility of separation congeneric species with molecular method.