The Calliphoridae include some of the most economically significant myiasis-causing flies in the world – blowflies and screwworm flies – with many being notorious for their parasitism of livestock. However, despite more than 50 years of research, key taxonomic relationships within the family remain unresolved. This study utilizes nucleotide sequence data from the protein-coding genes COX1 (mitochondrial) and EF1α (nuclear), and the 28S rRNA (nuclear) gene, from 57 blowfly taxa to improve resolution of key evolutionary relationships within the family Calliphoridae. Bayesian phylogenetic inference was carried out for each single-gene data set, demonstrating significant topological difference between the three gene trees. Nevertheless, all gene trees supported a Calliphorinae-Luciliinae subfamily sister-lineage, with respect to Chrysomyinae. In addition, this study also elucidates the taxonomic and evolutionary status of several less well-studied groups, including the genus Bengalia (either within Calliphoridae or as a separate sister-family), genus Onesia (as a sister-genera to, or sub-genera within, Calliphora), genus Dyscritomyia and Lucilia bufonivora, a specialised parasite of frogs and toads. The occurrence of cross-species hybridisation within Calliphoridae is also further explored, focusing on the two economically significant species Lucilia cuprina and Lucilia sericata. In summary, this study represents the most comprehensive molecular phylogenetic analysis of family Calliphoridae undertaken to date.