The genus Guettarda (Guettardeae–Rubiaceae) comprises approximately 150 species, ranging from eastern Africa through the islands of the Indian and Pacific Oceans to the Neotropics. Sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region were used to test the monophyly of Guettarda and its relationships to closely related genera within Guettardeae. The results indicate that Guettarda and two smaller genera, Antirhea and Stenostomum, are polyphyletic. Most Guettarda species fall into two distinct groups: a Neotropical lineage that also includes the widespread Indo-Pacific strand species G. speciosa (the type of the genus), and a New Caledonian lineage that, along with Antirhea and Timonius, comprises a dioecious Paleotropical clade. The Hawaiian endemic Bobea, traditionally considered close to Timonius and assumed to be of Old World origin, appears to be more closely related to Neotropical Guettarda species, suggesting that dioecy may have evolved twice within the tribe. The use of traditional gynoecium characters to delimit genera within Guettardeae is not congruent with the ITS phylogeny; other features, such as inflorescence architecture, sexual system, and palynology, appear to correlate more closely with the molecular phylogeny.