Despite extensive efforts, there are unresolved questions on evolutionary relationships in the angiosperm family Rubiaceae. Here, information from six loci and 149 Rubiaceae taxa provide new insights. Acranthera and Coptosapelta are strongly supported as sisters. Pollen grains of Acranthera possess several features common in Rubiaceae, but amongst potential similarities with the unusual grains of Coptosapelta are the nature of the apertures and the structure of the sexine. Luculia, Acranthera and Coptosapelta are excluded from the three subfamilies
Ixoroideae, Cinchonoideae and Rubioideae. Sipaneeae and
Condamineeae form a clade, sister to remaining Ixoroideae.
Rondeletieae and Guettardeae are sisters to remaining
Cinchonoideae. Colletoecema is sister to remaining Rubioideae, followed by the Urophylleae–Ophiorrhizeae clade. Nuclear ITS provided structured information at all phylogenetic levels, but the main gain from adding nrITS was the increased resolution. Average support values also increased but were generally high also without nrITS and the increase was not statistically significant.