Introduction: The aim of the present study was to evaluate the frequency of dentinal microcracks observed after root canal preparation with HyFlex CM (CoItene/Whaledent, Altstatten, Switzerland), HyFlex EDM (Coltene/Whaledent), Vortex Blue (Dentsply Tulsa Dental Specialties, Tulsa, OK), and TRUShape (Dentsply Tulsa Dental Specialties) systems using micro-computed tomographic (micro-CT) analysis. Methods: Forty human mandibular incisors with 1 and straight root canals were randomly assigned to 4 experimental groups (n = 10) and 1 control group for root canal preparation: group 1, HyFlex CM; group 2, HyFlex EDM; group 3, Vortex Blue; and group 4, TRUShape. The specimens were scanned using high-resolution micro-CT imaging before and after root canal preparation. Afterward, preoperative and postoperative cross-sectional images of the teeth were screened to identify the presence of dentinal defects. The number of microcracks was determined as a percentage for each group. Results: Before and after canal preparation, 36,152 cross-sectional images were examined. Four thousand four hundred fifty-two (12.31%) dentinal defects were observed. No new microcracks were observed after root canal instrumentation with the tested systems. Conclusions: Root canal preparation with the HyFlex CM, HyFlex EDM, Vortex Blue, and TRUShape systems did not induce the formation of new dentinal microcracks on straight root canals of mandibular incisors.