Background: Aim of this study was to analyze the root canal configuration in mandibular central and lateral incisors in vivo using cone-beam computed tomography (CBCT) imaging. Material and Methods: A total of 487 mandibular central incisors and 491 mandibular lateral incisors from 250 patients were examined using CBCT imaging, previously taken for diagnosis and treatment. The number of roots, root canal system configuration, presence of apical confluences, distance between confluences and radiographic root end, symmetry between left and right elements were recorded and statistically analyzed. Results: All the examined teeth presented only one root. No significant differences were found between the prevalence of two root canals in mandibular central incisors (219 teeth, 45%) compared to lateral incisors (211 teeth, 43%). Conclusions: The percentage of Vertucci type II configuration was higher than expected, being more frequent than type III. Type I was the prevalent, while other configurations were present but rare.