In this study, foliar and petiole anatomy of several genera was compared to determine whether there are characteristics that can be used to reevaluate the circumscription and taxonomic position of Hamelieae (Rubiaceae). Our sample included a total of 36 species, which were sectioned using conventional embedding and staining methods. From these species, 23 represented six of the seven genera of Hamelieae sensu Robbrecht; Syringantha Standl. was included in order to reevaluate its putative inclusion within Hamelieae. For comparative purposes, the sample also included representative species of tribes Psychotrieae (Rubioideae), the Portlandia informal group (Cinchonoideae), and Gardenieae (Ixoroideae). Our results indicated that foliar and petiole anatomy contained taxonomic information that can be used in systematic studies. Members of Hamelieae shared the following characters: cuticle more than 3 µm, dorsiventral mesophyll, a single palisade parenchyma cell layer, loose spongy parenchyma, raphides, tannins, and vascular tissue of types I, II, or III in the midrib and petiole. Plocaniophyllon Brandegee is unique in having fibers associated with major and minor veins. The petiole vascular tissue has an open arc shape in all studied species except in Randia L., which has a closed cylinder. Hamelieae, Syringantha, and Psychotria L. have raphides, one layer of palisade parenchyma cells, and loose spongy parenchyma, while the other taxa have druses, two layers of palisade parenchyma cells, and compact spongy parenchyma. Bouvardia Salisb. is unique, showing both raphides and druses. Our results show that comparative foliar and petiole anatomy may provide additional characters to be considered in taxonomic and phylogenetic studies within Rubiaceae. There was anatomical support for the inclusion of Syringantha within Hamelieae, while the presence of raphides and the mesophyll attributes suggest an understanding of why Hamelieae was morphologically treated as a member of Rubioideae.