BACKGROUND The purpose of this study was to evaluate the prevalence of middle mesial canal in mandibular first molars using cone-beam computed tomography (CBCT). METHODS In this descriptive study, a total of 768 radiographs of mandibular first molars, 384 for males and 384 for females, taken at a private radiology center in Isfahan, were assessed for the presence of middle mesial canal based on the gender of the patients. All samples had been prepared by one CBCT machine. The images were evaluated by endodontists and radiologists. Then, the morphology of first molar mesial root canals was assessed by Vertucci classification, and the obtained results were analyzed by SPSS software using a Chi-square test. The comparison was considered statistically significant at P < 0.05. RESULTS Twenty-four samples (3.13%) had a middle mesial canal. The prevalence rates of the middle mesial canal in the females and males were found to be 9 and 15 (2.35% vs. 3.92%), respectively, indicating no statistically significant difference (P = 0.21). Of 24 teeth with middle mesial canal, 17 samples (70.8%) were of Type XII and 7 samples (29.2%) were of Type VIII. There was no statistically significant difference between genders with regard to the prevalence of different types of middle mesial canals (P = 0.19). CONCLUSIONS The prevalence of the middle mesial canal was rather low in this study, and it is necessary to detect additional canals in the patients under root canal treatment.