The success of endodontic treatment depends on the identification of all root canals so that they can be cleaned, shaped, and obturated. This study investigated internal morphology of maxillary first molars by 3 different methods: ex vivo, clinical, and cone beam computed tomography (CBCT) analysis. In all these different methods, the number of additional root canals and their locations, the number of foramina, and the frequency of canals that could or could not be negotiated were recorded. In the ex vivo study, 140 extracted maxillary first molars were evaluated. After canals were accessed and detected by using an operating microscope, the teeth with significant anatomic variances were cleared. In the clinical analysis, the records of 291 patients who had undergone endodontic treatment in a dental school during a 2-year period were used. In the CBCT analysis, 54 maxillary first molars were evaluated. The ex vivo assessment results showed a fourth canal frequency in 67.14% of the teeth, besides a tooth with 7 root canals (0.72%). Additional root canals were located in the mesiobuccal root in 92.85% of the teeth (17.35% could not be negotiated), and when they were present, 65.30% exhibited 1 foramen. Clinical assessment showed that 53.26%, 0.35%, and 0.35% of the teeth exhibited 4, 5, and 6 root canals, respectively. Additional root canals were located in this assessment in mesiobuccal root in 95.63% (27.50% could not be negotiated), and when they were present, 59.38% exhibited 1 foramen. CBCT results showed 2, 4, and 5 root canals in 1.85%, 37.05%, and 1.85% of the teeth, respectively. When present, additional canals showed 1 foramen in 90.90% of the teeth studied. This study demonstrated that operating microscope and CBCT have been important for locating and identifying root canals, and CBCT can be used as a good method for initial identification of maxillary first molar internal morphology.