Purpose: This study was performed to compare the ability of limited- and large-volume cone-beam computed tomography (CBCT) to display isthmuses in the apical root canals of mandibular molars. Materials and Methods: Forty human mandibular first molars with isthmuses in the apical 3 mm of mesial roots were scanned by micro-computed tomography (micro-CT), and their thickness, area, and length were recorded. The samples were examined using 2 CBCT systems, using the smallest voxels and field of view available for each device. The Mann-Whitney, Friedman, and Dunn multiple comparison tests were performed (α=0.05). Results: The 3D Accuitomo 170 and i-Cat devices detected 77.5% and 75.0% of isthmuses, respectively (P>0.05). For length measurements, there were significant differences between micro-CT and both 3D Accuitomo 170 and i-Cat (P<0.05). Conclusion Both CBCT systems performed similarly and did not detect isthmuses in the apical third in some cases. CBCT still does not equal the performance of micro-CT in isthmus detection, but it is nonetheless a valuable tool in endodontic practice.