The first molecular phylogenetic analysis of the agriculturally important parasitoid subfamily Phasiinae (Diptera: Tachinidae) is presented, estimated from 128 worldwide taxa (80 genera) and approximately 7.6 kilobases of nuclear data. Special emphasis is placed on taxa with controversial taxonomic placement. The resultant phylogenetic tree is used to reconstruct ancestral character states, trace the evolution of significant adaptive traits within the Tachinidae, and test hypotheses about the classification of Phasiinae. Subfamily placements of the taxa Eutherini, Epigrimyiini, Litophasia, Strongygastrini, and Parerigonini are confidently resolved, the former three within Dexiinae and the latter two within Phasiinae. Due to sparse molecular evidence, the Imitomyiini are tentatively placed among the Phasiinae. Ancestral state reconstruction suggests a dominant and persistent trend in Phasiinae to evolve piercing structures used to insert eggs directly into host tissues. A single potential synapomorphy of Phasiinae is identified (elongated hypandrium). This phylogeny is used to update classification of worldwide phasiine genera and tribes. Many novel phylogenetic hypotheses are presented including the division of Parerigonini s. l. into three tribes: Parerigonini s. s., Zitini, and Cylindromyiini, and the division of Phasiini s. l. into four lineages: Phasiini s. s., Gymnosomatini, Opesiini, and Xystini. Two tribes are resurrected (Opesiini and Xystini) and one new tribe is proposed (Zitini nomen novum). Additionally, a survey of phasiine biodiversity was conducted in Great Smoky Mountains National Park (TN, NC). Species identifications were made using morphological keys, with further evidence from 900 base pairs of the nuclear coding gene MCS. In total, 221 specimens representing 26 phasiine species were collected. Of these, 21 species are newly recorded from the park, four are new records for Tennessee, and two are new records for North Carolina. All 12 eastern Nearctic phasiine genera were represented. Updated identification keys to eastern Phasiinae are provided and DNA barcoding sequences were generated that will aid future researchers to quickly and inexpensively identify phasiine species.