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Objective To analyze the morphology and incidence of middle mesial canal (MM) and isthmus in the mandibular first permanent molar by cone⁃beam computed tomography (CBCT). Methods Statistical analysis was per⁃ formed on images from patients who underwent CBCT examination in the Department of Radiology at Stomatological Hospital, Southern Medical University. Samples exhibiting root canal treatment, root resorption and calcification were ex⁃ cluded. Data regarding sex, age, the presence or absence of isthmus and MM, and the number of roots and root canals were recorded. Results Of the 217 mandibular first molar samples, 8 (3.7%) had an MM, and 2 (0.9%) had an inde⁃ pendent apical foramen. The overall incidence rate of isthmus was 57.1%; this rate was 50.7% in the cervical third of the root canal, 17.5% in the middle third, and 13.4% in the apical third. The incidence rate of isthmus was 61.8% on the left side, 52.3% on the right side, 58.7% in males, and 55.8% in females. No significant difference was found be⁃ tween the left and right sides or between females and males (P > 0.05). The incidence rate of isthmus in people under 60 was greater than 50% but was significantly lower in people older than 60. Conclusion Only a very small propor⁃ tion of MMs have an independent apical foramen in the mandibular first molar. The incidence of isthmus in the mesial root of the mandibular first permanent molar is high, and isthmus usually occurs in the cervical third of the root canal. During root canal treatment or apical surgery, attention should be given to the physical and chemical preparation of the isthmus.