Objectives The present study aimed to evaluate the frequency, position, and length of radicular pulp calcifications in three-rooted maxillary first molars using micro-computed tomography. Materials and methods Two hundred three-rooted maxillary first molars were scanned at a pixel size of 10 mu m and reconstructed using similar parameters. Three-dimensional models of the mesiobuccal (MB1, MB2, MB3), distobuccal (DB), and palatal (P) canals were qualitatively evaluated for discontinuity in the canal path. Transaxial and sagittal cross-sectional images of the roots were further screened, and the complete obliteration of the canal lumen by pulp calcification was identified when the canal had similar radiodensity to the surrounding dentine. Then, the number, position, and length of the calcifications were recorded for each root. Intra-observer agreement was performed using Cohen's kappa test, while one-way ANOVA and Duncan tests compared the lengths of calcifications among the canals, with alpha = 5%. Results Intra-observer agreement was perfect for identification of pulp calcifications (kappa = 1.0; p = 0.000). MB2 and MB3 canals accounted for the highest percentage frequency of calcifications distributed throughout the canal path. In the MB1, DB, and P canals, they occurred mainly at the apical third. Lengths of calcifications were greater in the MB3 (0.89 +/- 0.81 mm) and MB2 (0.82 +/- 0.93 mm) canals than in the MB1 (0.39 +/- 0.32 mm), DB (0.34 +/- 0.22 mm), and P (0.28 +/- 0.22 mm) canals (p < 0.05). Conclusion MB2 and MB3 canals accounted for the highest frequency and length of radicular pulp calcifications. In the MB1, DB, and P canals, calcifications occurred mostly at the apical third, while in the MB2 and MB3, most of calcifications were observed at the orifice level and along the canal path.