Introduction: The aim of this investigation was to evaluate the effect of the presence and preparation of middle mesial (MM) canals on the fracture resistance of the mesial root of mandibular molars. Methods: Forty intact mesial roots of mandibular first molars having 2 (n = 20) or 3 (n = 20) independent canals from the furcation level for up to at least 5 mm apically were selected based on preoperative micro-computed tomographic scanning. The selected roots were then distributed into 2 experimental (n = 10) and 2 control groups (n = 10) according to the root length, canal configuration (2 or 3 independent canals), and root thickness at the furcation level. In the experimental groups 1 (2 independent canals) and 3 (3 independent canals), root canals were enlarged up to the ProTaper Next X3 rotary instrument (Dentsply Sirona, Ballaigues, Switzerland), whereas in groups 2 (2 independent canals) and 4 (3 independent canals) root canals were not prepared. The specimens were embedded in acrylic resin after their surfaces were coated with a thin layer of silicone and subjected to a fracture strength using a universal testing machine. The types of fracture extension and course were recorded and statistically compared with the chi-square test, whereas fracture strength was analyzed using 1-way analysis of variance and post hoc Tukey tests (alpha = 5%). Results: No statistical difference in fracture strength was observed between nonprepared roots with 2 (group 2, 696.1 +/- 186.3 N) or 3 (group 4, 558.4 +/- 154.6 N) independent canals (P > .05), whereas the lowest values were obtained in the prepared roots with an MM canal (groups 3, 377.1 +/- 77.2 N) (P < .05). The mean fracture strength observed in the prepared roots with 2 canals (group 1, 528.4 +/- 134.3 N) showed no statistically significant difference compared with nonprepared roots with 3 canals (group 4, 558.4 +/- 154.6 N) (P > .05). The chi-square test revealed no significant differences in fracture extent, types, and courses among groups (P > .05). Fracture extensions in all groups were mostly central and buccalcentral types, whereas the highest frequency of fracture course was the curved and zigzag types. Conclusions: The fracture resistance of the mesial roots of the mandibular molars decreased after the preparation of mesial canals with large-tapered instruments. Preparation of the MM canal further diminished the fracture resistance of the mesial roots. The resultant fracture displayed a distinct pattern in the buccolingual plane.