Objective: This study was conducted to determine the most prevalent root canal configuration and the incidence of a second mesio-buccal (MB2) canal in maxillary first molar root in an Indian sub-population of Pune, Maharashtra, India by reviewing cone-beam computed tomographic (CBCT) images. Methods: Patients who had undergone CBCT scanning for various treatment modalities were retrospectively viewed for the presence of second MB2 canal in the permanent maxillary first molar. Scanning was performed at Peraden & Elite CBCT, Pune, Maharashtra, India. Each tooth was assessed in 1 mm axial and 0.5 mm sagittal sections, to evaluate the presence of a MB2 canal. The images had been evaluated by a maxillofacial radiologist. Results: In total of 117 teeth examined, MB-2 was present in 79 teeth. The prevalence of MB-2 in maxillary first molar was found to be 67.5% in an Indian subpopulation. As far as root canal morphology is concerned, Vertucci Type I was the most common type. Conclusion: Knowing the incidence of presence of MB2 canal in the first maxillary permanent molar is very essential for the success of the root canal treatment & CBCT is very vital to serve this purpose.