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Introduction: Failures of root canal treatments are mainly attributed to missed canals and ignorance about the complex anatomy of the root canal system. One such example of anatomic variation is the middle mesial canal (MMC) in mandibular molars which is often missed, and literature on methods for identifying them is limited. Aim: This in vitro study aimed to assess the occurrence of MMCs with cone-beam computed tomography (CBCT) and then under magnification in the mandibular first and second molars. Materials and Methodology: A total of 120 extracted intact human permanent mandibular first and second molars were selected. These were subjected to CBCT imaging and magnification for the detection of MMCs. The percentage of incidence in the detection of MMC was compared between these two methods. Results: The incidence of MMC detected in mandibular molars using CBCT was 13.33% (16 of 120 teeth), while using a dental operating microscope, the incidence was 18.33% (22 of 120 teeth) and the comparison between the two methods was statistically not significant. The MMCs were classified based on the Pomeranz Classification. The most prevalent canal configuration was a confluent type which was found to be in 72% cases (16 of 22), followed by fin type which was 22.7% (5 of 22) and independent type was only 4.54% (1 of 22). The incidence of the location of MMC orifice was studied. In 50% of the cases, orifice was located closer to the mesiolingual canal, whereas in 27% of the cases, it was closer to the mesiobuccal canal and in 4.95% of the cases, it was detected midway. Conclusions: Since the incidence of MMC was higher with the use of a microscope, it is preferred to use simpler, chairside aids like magnification and ultrasonic troughing. On the other hand, one should be judicious while subjecting a case to preoperative CBCT evaluation owing to its ionizing radiation.