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Objective To investigate and analyze the root and root canal morphology of mandibular first molars (MFMs) in the Tibetan population using cone-beam computed tomography (CBCT) and to provide references for clinical root canal treatment in the Tibetan population. Methods This study was reviewed and approved by the Ethics Committee, and informed consent was obtained from the patients. CBCT imaging data of 300 mandibular first molars from 300 Tibetan patients were included. Patient age, the number of roots in mandibular first molars were recorded. The morphology and incidence of mesial root and mesial root canals and the morphology and incidence of distal root and distal root canals were statistically analyzed by Vertucci classification. Results There were 198 double-root teeth and 102 three-root teeth in the 300 mandibular first permanent molars. Among the three-rooted molars, 1 case had mesiolingual roots, and the rest had distolingual roots. The incidence rate of the distolingual root was 33.7%(101/300). The most common root canal configuration was Vertucci Ⅳ 65.7% (197/300), followed by Vertucci Ⅱ 20.3% (61/300) in the mesial roots. The overall incidence of middle mesial canals (MMCs) was 6% (18/300), with the highest incidence of MMCs in the 20-40 year-old group at 9% (9/100). The distal roots canals of single-distal-rooted mandibular first molars were mainly Vertucci Ⅰ 66.8% (133/199), followed by Vertucci Ⅱ 14.6% (29/199) and Vertucci Ⅳ 11.6% (23/199). For the mandibular first permanent molars with two distal roots, 96% (97/101) of the distal buccal roots and 100% (101/101) of the distal lingual roots were Vertucci Ⅰ root canals. Conclusion The root and root canal morphology of mandibular first permanent molars in a Tibetan population is complex and variable. Approximately one-third of patients have distolingual roots, and clinicians should carefully explore the root canals under the guidance of CBCT.