Objective: The aim of this study was to investigate in vivo, using cone beam computed tomography (CBCT), the differences between genders regarding the number of roots and root canal morphology. Method and Materials: An existing CBCT scan database was accessed and the scans were divided in two groups according to gender. Tooth inclusion and exclusion criteria were applied. The examined teeth were classified according to the number of roots, and the root canal system was classified according to Vertucci classification. Z-test for independent groups was used to detect differences between groups. Results: In total, 12,325 teeth (4,597 from males and 7,728 from females) from 670 patients were analyzed. Females presented a lower number of roots per tooth in all groups of teeth, except for mandibular canine, the difference being statistically significant in the maxillary first premolar and both maxillary and mandibular second molars. Vertucci Type I configuration had a higher or equal prevalence in nine of the eleven groups of maxillary roots in females, with significance in both maxillary premolars. The same configuration was higher in females in five of the nine groups of mandibular roots, with significance in mandibular central incisor and first premolar. Eight of the nine root groups (maxillary and mandibular roots combined) that presented three-root canal system configurations, had a higher prevalence of that anatomy in males. Conclusion: Few differences were found between genders. It was possible to detect a lower number of roots per tooth and a higher number of Vertucci Type I configurations in females. The three-root canal system configurations were more common in males. Keywords: anatomy, cone beam computed tomography, gender, morphology, root canal