Introduction: For appropriate root canal therapy, understanding the anatomical and morphological aspects of the endodontic area is critical. Therefore, the study and analysis of images is an important step in the assessment and current clinical treatment of endodontic problems. Furthermore, microscopic-assisted endodontic therapy, particularly for multirooted teeth, is becoming more common in contemporary endodontic clinical practice. The aim of this in vitro study was to examine the distinct types of isthmuses using a light microscope and Cone-Beam Computed Tomography (CBCT). Method: Fifty-five extracted human molars were used for this study. CBCT was used to realize the study. After the CBCT analysis, all teeth were inspected under a light microscope, and the results were compared with those of the CBCT examination. Results: The results from the CBCT at the coronary third of the root canals were as follows: Types I, II, III, IV, and V isthmuses were found in 50.9%, 16.4%, 1.8%, 7.3%, and 23.6% of all samples, respectively. For the middle third, we found Types I, II, III, IV, and V isthmuses in 40.0%, 23.6%, 1.8%, 16.4%, and 18.2% of the samples, respectively. For the apical third, we found Types I, II, III, IV, and V isthmuses in 60.0%, 29.1%, 5.5%, 1.8%, and 3.6% of all samples, respectively. The results obtained from the microscopic examination were similar to those from the CBCT study. There is no confirmed statistically significant difference between the examination methods (p = 0.07). Conclusion: Three-dimensional examinations have a significant role among the methods for diagnosing and detecting an isthmus in the complex root canal system, especially in the apical part.