A comprehensive tribal-level classification for the world's subfamilies of Hesperiidae, the skipper butterflies, is proposed for the first time. Phylogenetic relationships between tribes and subfamilies are inferred using DNA sequence data from three gene regions: 943 bp of cytochrome oxidase subunit I (COI) (in the mitochondrial genome), 739 bp of elongation factor-lα (EF-lα) and 403 bp of wingless (both in the nuclear genome). Morphological character scores were then added to the molecular matrix, for a combined simultaneous analysis. In both analyses, branch support for each clade is investigated, and conflict among data partitions is assessed using the partition congruence index. Monophyly of the family Hesperiidae is strongly supported, as are some of the traditionally recognized subfamilies, with the following relationships (from the combined molecular and morphological analysis): (Coeliadinae + (Euschemoninae + (Eudaminae + (Pyrginae + (Trapezitinae + (Heteropterinae + Hesperiinae)))))). The formerly recognized subfamily Pyrrhopyginae, while strongly supported as a monophyletic group, is downgraded to a tribe of Pyrginae. The former subfamily Megathyminae is considered to be an infra-tribal group of Hesperiinae (Erionotini), although its phylogenetic position therein remains uncertain. The Australian endemic Euschemon rafflesia is a hesperiid, given subfamily-status. Most of the traditionally recognized generic groups and subgroups currently employed to partition the subfamilies of Hesperiidae are not monophyletic. Seven subfamilies of Hesperiidae are recognized: Coeliadinae, Euschemoninae (confirmed reinstated status), Eudaminae (new status), Pyrginae, Trapezitinae, Heteropterinae (confirmed status) and Hesperiinae. Pyrrhopygini (reinstated status), Tagiadini (confirmed reinstated status), Celaenorrhinini (confirmed new status), Carcharodini (reinstated status), Achlyodini (new status), Erynnini (confirmed new status) and Pyrgini (confirmed status) are treated as tribes of Pyrginae, but the circumscription of Achlyodini and Pyrgini requires further elaboration. Tribes of Hesperiinae include Aeromachini (new status), Erionotini (new status), Baorini (new status), Taractrocerini (confirmed reinstated status), Thymelicini (confirmed reinstated status), Calpodini (reinstated status), Anthoptini (new tribe), Moncini (new tribe), and Hesperiini (confirmed reinstated status), but the circumscription of Erionotini should be considered tentative. / Graduation date: 2006