Introduction The purpose of this study was to quantitatively analyze the root canal morphology of three-rooted mandibular first molars by micro–computed tomography (micro-CT) scans. Methods Twenty three-rooted mandibular first molars were scanned by micro-CT scans. The teeth were reconstructed three-dimensionally by software Mimics 10.01 (Materialise, Leuven, Belgium). The levels of the root apexes and furcations; the diameters; wall thicknesses; and tapers of the mesiobuccal (MB), mesiodistal (ML), distobuccal (DB) and distolingual (DL) canals were determined. The buccolingual/mesiodiatal (BL/MD) diameter ratios (ΔC) and the prevalence of long oval canals (ΔC > 2) at different levels were calculated. Results The level of distal furcations was significantly ( P < .01) lower than that of MD furcations with an average of 1.09 mm. The buccal and lingual walls were significantly ( P < .05) thicker than the mesial and distal for the MB, ML, and DB canals. Among four canals, the DL canal had the smallest mean value of ΔC at each level. The average BL taper was greater than the MD for each canal except the middle portion of the DL canal. Conclusion The vertical length, width, BL/MD diameter ratio, wall thickness, and BL taper of the DB canals are on average greater than the DL canals. The MB, ML, and DB canals are more oval, whereas the DL canals are relatively rounder. The geometric data of root canals are useful for endodontic treatment on three-rooted mandibular first molars.