This study aimed to evaluate the spatial location of the second mesiobuccal canal (MB2) of maxillary molars (MMs), using cone-beam computed tomography (CBCT), in a Brazilian subpopulation. The CBCT examination of 250 patients performed on the Eagle 3D device was analyzed, totaling 787 MMs. Using the Radiant Dicom Viewer software, measurements were made of the distances, in millimeters (mm), between the first mesiobuccal canal (MB1), MB2, and palatal (P) canal inputs, from the axial sections. The Image J software evaluated the angle formed by the lines. The data obtained were analyzed statistically by Fisher's exact and Chi-square tests with a significance of 5%. The prevalence of MB2 canals observed was 76.44 and 41.73% in the first and second molars (1MMs and 2MMs), respectively (p < 0.05). The average of the distances and angles performed, for the location of the MB2 canals of the analyzed teeth, were MB1-P = 5.83 mm, MB1-MB2 = 2.31 mm, and MB2 for the intersection of the connecting distance from MB2-T = 0.90 mm. The average angle formed between the MB1-P and MB1-MB2 distances was 25.89° and 19.68° for the 1MMs and 2MMs, respectively. It was also observed that 91.4 and 75.4% of the maxillary 1MMs and 2MMs, respectively, presented the MB2 canals mesially located at the line connecting the MB1-P canals (p < 0.0001). The MB2 canals were located mesially to the MB1 canal with an average distance of 2 mm between the canals. The anatomical knowledge of the spatial location of the MB2 canal in different ethnicities is important for the planning and execution of endodontic treatment.