In the present study, a molecular phylogeny of the Rondeletia L. complex (Rubiaceae, Rondeletieae) was constructed with the following main objectives: (1) to evaluate the sections of Rondeletia proposed by Fernández Zequeira; (2) to test if Stevensia Poit. belongs to the Rondeletieae s. str.; (3) to check if ITS data from Rondeletieae support previous phylogenetic results from trnL-F data regarding circumscription of Rondeletieae; and (4) to verify if Hodgkinsonia F. Muell. belongs to Guettardeae or elsewhere. Two analyses were performed, one with ITS sequences from 46 taxa in the Rondeletieae–Guettardeae complex, and the other with combined ITS, rps16, and trnL-F sequences of 21 taxa. Representatives of nine of the 10 sections of Rondeletia recognized by Fernández Zequeira were included in the ITS analysis. Five of her sections could be tested for monophyly. Support was only found for Rondeletia sect. Leoninae M. Fernández Zeq., while representatives from section Chamaebuxifoliae M. Fernández Zeq., section Hypoleucae Standl., and section Nipenses M. Fernández Zeq. together form a well-supported clade that could be distinguished also based on morphology. The latter clade is sister to Stevensia, which is thus placed within Rondeletia s. str. In addition, ITS sequence data confirm the separation of Rovaeanthus Borhidi from Rogiera Planch. Support is low for inclusion of Blepharidium Standl., Mazaea Krug & Urb., Phyllomelia Griseb., Rachicallis DC., part of Rogiera, and Suberanthus Borhidi & M. Fernández Zeq. in Rondeletieae. Rachicallis, Mazaea, and Phyllomelia form a clade with strong support. The tribe Rondeletieae s. str. was found to be monophyletic in all trees, although with low support; however, a re-delimitation of the tribe is proposed here based on this study and previous phylogenetic analyses. The monophyly for the tribe Guettardeae is weakly supported, with the inclusion of Arachnothryx Planch. (including Cuatrecasasiodendron Steyerm.), Gonzalagunia Ruiz & Pav., Hodgkinsonia, and Timonius DC. Although it was recently the subject of a molecular phylogenetic study, the tribe Guettardeae is still in need of a wide-ranging survey in order to confirm its monophyly and delimit its taxonomic boundaries. Because Cuatrecasasiodendron was found within the Arachnothryx clade, the two genera are here synonymized as Arachnothryx, and in turn positioned within the tribe Guettardeae. In addition, based on herbarium and field studies, the two species described under Cuatrecasasiodendron (C. spectabile Steyerm. and C. colombianum Standl. & Steyerm.) are treated as synonyms to the new combination Arachnothryx spectabilis (Steyerm.) Rova, Delprete & B. Bremer, which