The current study aimed to investigate the root canal anatomy and possible variations in permanent mandibular incisor teeth (central and lateral) of an Indian subpopulation, using three-dimensional cone-beam computed tomography (CBCT) imaging in vitro. Material and Methods: A total of 200 freshly extracted permanent mandibular incisors (100 central incisors and 100 lateral incisors) were collected from dental hospitals and private clinics in Mumbai and Navi Mumbai cities from the state of Maharashtra. Teeth with fully formed apices, no calcifications, no previous endodontic treatment, absence of root fracture, absence of internal/external resorption were included in the study. The collected samples were exposed to CBCT scanning and the root canal anatomy with variations (if any) was tabulated according to Vertucci's classification for each sample. Results: Of the 200 teeth analyzed the most common root canal configuration was Type I (55.5%), followed by Type III (24.14%), Type II (12.08%), and Type IV (3%). Also, of the 111 Type I canals, 60.37% (67 off 111) presented round, while 39.63% (44 of 111) presented non-round canals in the coronal and the middle thirds. At the apical third of the canals 81.08% (90 of 111) exhibited round, whereas 18.91% (21 of 111) exhibited non-round cross-section.