The project’s objectives were to collect reference adult dipteran specimens from across the 48 contiguous states, with a focus on specimens from widely spread geographic localities; to identify a genetic locus that is suitable for discrimination of relevant species in a phylogenetic analysis; and to develop a reference DNA database. The database will have associated with it all original physical specimens preserved as vouchers, with sufficient morphological features retained to enable future re-examination of the specimens. The research had a two-phase design. It first examined a range of genetic loci from a limited number of species and individuals, in order to assess the relative performance of the different loci (and regions therein) at discriminating among species. In the second phase, data were collected from a large number of species and individuals for the locus determined in the first phase to be best for discrimination among forensically important species. The project succeeded in collecting and identifying just over 2,500 flies from 84 counties in 20 states. The study’s first phase determined that any region of the mitochondria genome was likely to be sufficient for discrimination among most forensically relevant species, but that a region within the mitochondria encoded gene Cytochrome oxidase I was best for discrimination between a closely related pair of species. DNA sequence for the chosen locus was obtained for 504 individual flies representing 106 distinct species. The reference DNA database is suitable for correct species identification of 16 of the 18 species most important for forensic investigations.