Carrion-breeding Sarcophagidae (Diptera) can be used to estimate the post-mortem interval in forensic cases. Difficulties with accurate morphological identifications at any life stage and a lack of documented thermobiological profiles have limited their current usefulness. The molecular-based approach of DNA barcoding, which utilises a 648-bp fragment of the mitochondrial cytochrome oxidase subunit I gene, was evaluated in a pilot study for discrimination between 16 Australian sarcophagids. The current study comprehensively evaluated barcoding for a larger taxon set of 588 Australian sarcophagids. In total, 39 of the 84 known Australian species were represented by 580 specimens, which includes 92% of potentially forensically important species. A further eight specimens could not be identified, but were included nonetheless as six unidentifiable taxa. A neighbour-joining tree was generated and nucleotide sequence divergences were calculated. All species except Sarcophaga (Fergusonimyia) bancroftorum, known for high morphological variability, were resolved as monophyletic (99.2% of cases), with bootstrap support of 100. Excluding S. bancroftorum, the mean intraspecific and interspecific variation ranged from 1.12% and 2.81–11.23%, respectively, allowing for species discrimination. DNA barcoding was therefore validated as a suitable method for molecular identification of Australian Sarcophagidae, which will aid in the implementation of this fauna in forensic entomology.