Relationships among 16 taxa of Rondeletia complex (Rubiaceae) are estimated using nuclear ribosomal internal transcribed spacer 1 sequence (ITS1). Three species from three more distantly genera in Rondeletieae (one African, two Neotropical) were chosen as outgroup. Previous molecular studies of trnL-F and rps16 data showed that Rondeletia and Rondeletieae are polyphyletic. At present study an alternate set of species were included from Mexico and Central America, and ITS1 was introduced into the molecular analysis, as a new sequence. By analysing of ITS1 region, Arachnothryx, Rondeletia and Rogiera genera were well separated within Rondeletieae tribe.