OBJECTIVES: To assess the internal and external morphologies of the mesiobuccal (MB) root of maxillary molars presenting a third root canal (MB3), using micro-computed tomography (micro-CT). MATERIAL AND METHODS: Two-hundred and sixty-five extracted maxillary first and second molars with different root configurations were imaged in a micro-CT scanner at 19.6-μm pixel size. Sixteen teeth presenting MB3 canal were selected and evaluated regarding root configuration, minimal dentine thickness 2 mm under the furcation area, canal configuration of the MB root, MB3 canal morphology (location, independent or confluent orifice, and anatomy types), and the apical anatomy (aspect ratio, number of accessory canals and foramina, presence of isthmus, and independent MB3 foramen). RESULTS: Overall, a high variability in canal configuration was detected in the MB root. MB3 canal was observed in 10 maxillary first molars and 6 maxillary second molars (n = 16). Minimal dentine thickness related to the MB3 canal at the coronal third was smaller than that of the MB1 canal. A complex internal anatomy comprising 13 different root canal configurations was observed. A high number of independent MB3 orifices at the pulp chamber floor was observed in the first molars (7 out of 10 teeth), while most of the specimens (n = 14) showed a confluent anatomy of the MB3 canal. A varied number of accessory canals and foramina were observed. At the apical third, isthmus could be observed in 6 specimens, while an independent MB3 foramen was present in 37.5% of the MB roots. CONCLUSIONS: MB3 canal is a rare anatomical variation present in maxillary first and second molars. Its presence can be associated to a complex internal anatomy of the MB root which includes the presence of isthmuses and multiple accessory canals and foramina at the apical third, but also a thin dentine thickness at the coronal third and a confluent anatomy of the MB3 with the other main canals.