A new, monophyletic, genus-level classification of the Gentianaceae is presented based on cladistic analyses of trnL intron, matK, and internal transcribed spacer (ITS) sequence data (key pp. 45–47; conspectus pp. 48–55). The family as presently circumscribed contains 87 genera and c. 1615–1688 species. Our analyses include nucleotide sequence data for 66 genera, for which no attempt has been made to test their monophyly. Some genera that could not be represented by DNA data are placed in higher categories based on morphological considerations. Our phylogenetic results suggest the recognition of six tribes: Exaceae, Chironieae, Gentianeae, Helieae, Potalieae, and Saccifolieae. The tribe Saccifolieae (Maguire & Pires) Struwe, Thiv, V. A. Albert, & Kadereit, stat. nov. is the most basally positioned lineage in the family and consists of five neotropical genera: Curtia, Hockinia, Saccifolium, Tapeinostemon, and Voyriella. The Exaceae, which is the next-most basal clade in the Gentianaceae, is paleotropical and includes the following genera: Cotylanthera, Exacum, Gentianothamnus, Ornichia, Sebaea, and Tachiadenus. Following Exaceae is tribe Chironieae, divided into three subtribes (which are resolved as a trichotomy): Chironiinae, Canscorinae Thiv & Kadereit, subtrib. nov., and Coutoubeinae. Subtribe Chironiinae is pantropical to temperate and includes Bisgoeppertia, Blackstonia, Centaurium, Chironia, Cicendia, Eustoma, Exaculum, Geniostemon, Ixanthus, Orphium, Sabatia, and Zygostigma. The paleotropical subtribe Canscorinae contains Canscora, Cracosna, “Duplipetala”, Hoppea, Microrphium, Phyllocyclus, and Schinziella. The third subtribe in Chironieae, the Coutoubeinae, is strictly neotropical with Coutoubea, Deianira, Schultesia, Symphyllophyton, and Xestaea.