A phylogenetic analysis of 70 species of the tribe Rubieae and of 25 taxa belonging to 14 other tribes of Rubiaceae has been performed using the DNA sequence of the chloroplast atpB-rbcL intergene region. A characteristic 204-bp deletion is shared by all investigated taxa of the tribes Coccocypseleae, Psychotrieae, Hedyotideae, Spermacoceae, Anthospermeae, Theligoneae, Paederieae, Rubieae, and by Mitchella. This undoubtedly monophyletic group is nearly identical with the subfamily Rubioideae of Robbrecht (1988, 1993). Molecular data for Ophiorrhizeae, Hamelieae, and Hillieae suggest a position outside of this monophylum. An additional 50-bp deletion characterizes all investigated members of the tribe Rubieae (inclusive of Didymaea), which is, thus, also clearly shown to be monophyletic. The five Rubieae clades already recognized (Rubia, Asperula sect. Glabella, Sherardia, Cruciata, and Galium seer. Galium) are supported by additional sequences, the basal Didymaea clade and the Asperula sect. Asperula clade are new. Galium and Asperula appear to be of polyphyletic origin. Relbunium belongs to the Cruciata clade.