Subfamily Ixoroideae is one of three major lineages in Rubiaceae, with approximately 4000 species. Previous molecular phylogenetic studies have indicated that many genera and tribes previously placed in other subfamilies are better considered as part of Ixoroideae. However, the internal resolution and clade support have generally been low, and several genera found to be nested in the subfamily do not appear to be associated with any described tribe. In order to resolve the phylogeny and assess the tribal delimitations in the expanded Ixoroideae, phylogenetic reconstructions were performed using Bayesian and parsimony analyses of six plastid DNA regions and a broad sampling of genera from all tribes of the subfamily. In the inferred phylogenetic hypotheses, the tribal relationships were mostly well supported, with Ixoroideae consisting of the Coffeeae and the Vanguerieae alliances as sister groups and a grade comprising Condamineeae, Henriquezieae, Posoquerieae, Retiniphylleae, Sipaneeae and the genus Steenisia. A revised tribal classification, including the description of five new tribes, Airospermeae, Augusteae, Scyphiphoreae, Steenisieae and Trailliaedoxeae, is provided. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 387–406.