The most diverse and best-preserved early fauna of flies (order Diptera) is described from the Late Carnian (Late Triassic, ca. 220 Ma) of Virginia, USA. Complete flies are preserved as aluminosilicate films on very fine-grained shales from the Cow Branch Formation, which is part of the Newark Supergroup of Early Mesozoic rift basins from eastern North America. The dipteran fauna consists of eight families (one new), 11 genera (five new), and 16 species (11 new), and includes the following taxa (Blagoderov and Grimaldi are the authors of all new names): Architipula youngi Krzemiński, Metarchilimonia krzeminskorum n.gen., n.sp., and M. solita n.sp. (Limoniidae); Triassopsychoda olseni n.gen., n.sp. (Psychodidae); Culicomorpha indet.; Yalea argentata (Krzemiński), Y. rectimedia n.sp., Alinka cara Krzemiński (Procramptonomyiidae); Veriplecia rugosa n.sp., Virginiptera certa n.gen., n.sp., V. similis n.sp., V. lativentra n.sp. (Paraxymyiidae); Brachyrhyphus distortus n.gen. n.sp. (Protorhyphidae); ?Crosaphis virginiensis n.sp. (Crosaphididae); and Prosechamyia trimedia n.gen., n.sp., P. dimedia n.sp. (Prosechamyiidae, new family). Particularly significant is a culicomorphan with a long proboscis, which is the earliest fossil record of a structure specialized apparently for blood feeding. Also, Prosechamyia appears to be a stem group to the very diverse infraorder Brachycera, the earliest definitive members of which appear in the Early Jurassic. Phylogenetic relationships of major clades of living and extinct nematocerous Diptera are analyzed, indicating that infraordinal-level diversification was complete by the Late Triassic. Flies did not reach modern levels of ecological abundance until the mid-Jurassic, apparently due to diversification within most infraorders by that time.