Cone-beam computed tomography (CBCT) can provide valuable data for root canal systems of human teeth. This study used CBCT to evaluate the number of roots and canals in mandibular first molars in Taiwanese individuals. We screened 151 patients (76 male and 75 female) scheduled for CBCT scan prior to implantation, or orthodontic, endodontic, or periodontic treatment between June 2006 and March 2009 at the Department of Dentistry, Cardinal Tien Hospital, Taipei, Taiwan. A total of 237 image samples of mandibular first molars were obtained and analyzed for their number of roots and canals. We found that 177 (74.7%) mandibular first molars had two roots and 60 (25.3%) had three roots. Of the 237 teeth, 133 (56.1%) had three canals, 96 (40.5%) had four, and eight (3.4%) had two canals. Of the 86 patients (43 male and 43 female) with bilateral mandibular first molars, 22 (25.6%, 15 male and 7 female) had bilateral three-rooted mandibular first molars, and six (7.0%, 4 male and 2 female) had unilateral three-rooted mandibular first molars. The chi(2) test showed a significantly higher incidence of three-rooted mandibular first molars in male (44.2%, 19/43) than in female (20.9%, 9/43, p = 0.038) subjects. Our results showed a high overall incidence (32.6%) of three-rooted mandibular first molars in Taiwanese individuals. CBCT could be a valuable tool for identifying an extra distolingual root in mandibular first molars.