The purpose of this study was to investigate the three-dimensional (3-D) morphology of isthmuses in the mesial roots of mandibular molars.One hundred twenty-six extracted mandibular molars including 70 first molars (MFMs) and 56 second molars (MSMs) from a Chinese population were scanned by micro–computed tomography, and the apical 5 mm of the root canals with or without isthmuses was reconstructed with 3D-Doctor software. Each reconstructed isthmus model was assigned to 1 of the following 4 categories: sheet connection, separate, mix, and cannular connection. The length of the isthmus (L i ) and the distance from the bottom of the isthmus to apex (D ba ) were measured and analyzed.The incidence of an isthmus in the apical 5 mm of the mesial roots was 85%. MFMs had more isthmuses with separate type and mixed type, whereas MSMs had more isthmuses with sheet connections. The L i and D ba in mandibular molars were 2.331 mm and 1.850 mm, respectively.MFMs present different anatomical features of isthmuses in the apical part of the mesial roots than MSMs.