The mandibular first molars mostly have two mesial canals. In this report, two cases of mandibular first molars with three mesial canals are presented. The middle mesial canal was detected under endodontic microscope and further confirmed by cone-beam computed tomography and angled radiography, respectively. The purpose of this paper was to highlight the possibility of an aberrant root canal in a mandibular first molar and to help reduce the failure rates of the dental pulp treatment of the mandibular first molar.