Objectives: To determine the prevalence of Middle Mesial Root Canal (MMC) in a Brazilian subpopulation, verify whether its presence is related to anatomical characteristics of the tooth, and propose a classification using cone-beam computed tomography (CBCT). Methods: CBCT scans of 284 patients were evaluated by two radiologists to determine the presence of the MMC in mandibular first and second molars. Subsequently, the mesio-distal and buccal-lingual measurements of the mesial roots were obtained; the measurements between the root canals, and from MMC to the mesiobuccal canal and to the mesiolingual canal were also obtained. The MMC was classified according to its relationship with mesiobuccal and mesiolingual canals. The data were analyzed using chi-square and Fisher’s exact test, Multiple Logistic Regression analysis, Student’s t-test, κ and Intraclass correlation coefficient (p < 0.05). Results: The intra examiner agreements for the presence of the MMC and the measurements were considered almost perfect (0.953 and 0.999, respectively). Of 216 mandibular first molars, 11.1% had the MMC, and of 228 mandibular second molars, only 1.75% had the MMC. The presence was significant higher in the mandibular first molar (p < 0.0001). The buccal-lingual measurement and the measurement between mesiobuccal and mesiolingual canals were higher in teeth with MMC (p = 0.024 and p = 0.005, respectively). It was possible to classify the configuration of MCC into four types: Independent (60.7%) Confluent (14.3%), Mesiolingual confluent (14.3%), and Mesiobuccal confluent (10.7%). Conclusions: The prevalence of MMC is more pronounced in mandibular first molars and anatomical measurements, such as greater measurement between mesiobuccal and mesiolingual canals, may alert clinicians to its presence. It was found 4 types of configurations of MMC.