show Abstracthide AbstractWe obtained and analyzed whole genome data for more than 160 representatives of Skipper butterflies (family Hesperiidae) from all known subfamilies, tribes and most distinctive genera. We find that two genera, Katreus Watson, 1893 and Ortholexis Karsch, 1895, which are sisters, are well-separated from all other major phylogenetic groups and originate near the base of the Hesperiidae tree, prior to the origin of some subfamilies. Due to this ancient origin compared to other subfamilies, this group is described as Katreinae Grishin, subfam. n. DNA sequencing of primary type specimens reveals that Ortholexis melichroptera Karsch, 1895 is not a female of Ortholexis holocausta Mabille, 1891, but instead a female of Ortholexis dimidia Holland, 1896. This finding establishes O. dimidia as a junior subjective synonym of O. melichroptera. Furthermore, we see that Chamunda Evans, 1949 does not originate within Pyrginae Burmeister, 1878, but, unexpectedly, forms an ancient lineage of its own at the subfamily rank: Chamundinae Grishin, subfam. n. Finally, the group of two sister genera, Barca de Nicéville, 1902 and Apostictopterus Leech, [1893], originates around the time Trapezitinae Waterhouse & Lyell, 1914 have split from Hesperiinae Latreille, 1809. A new subfamily Barcinae Grishin, subfam. n. consisting of Barca and Apostictopterus sets them apart from all other Hesperiidae.