Background: One of the main reasons for the failure of root canal treatment is the incomplete knowledge of the root canal system. With respect to the complexity of maxillary molars root canal system, and the possibility of the relationship between the buccolingual width of the mesiobuccal root and root canal morphology in maxillary molars, the aim of this study is to determine this relationship with cone-beam computed tomography (CBCT). Materials and Methods: This in vitro study carried out on 311 CBCT scans. Maxillary first molars ( n = 311) were evaluated in three sagittal, axial, and coronal planes. For each tooth the number of canals, presence of second mesiobuccal (MB2), buccolingual width of mesiobuccal root at the cementoenamel junction (CEJ), and mid-root level, and type of canals according to the Vertucci's classification were determined. Results: The results showed that 49.1% of first maxillary molars had 3 and 50.8% had four root canals. The most common canal type in the mesiobuccal root was Vertucci's Type I followed by Types II, IV, and V. The prevalence of MB2 in the Iranian subpopulation was 50.1%. In maxillary first molars with MB2, the buccolingual width of mesiobuccal root at the CEJ and mid-root level ( P < 0.001) was significantly greater than the corresponding values in other one without MB2. Conclusion: The results showed that the buccolingual width of mesiobuccal root in maxillary first molars at the CEJ level and mid-root was correlated with the number of root canals.