© 2017 American Association of EndodontistsIntroduction The aim of the present study was to evaluate the frequency of dentinal microcracks observed after root canal preparation with HyFlex CM (Coltène/Whaledent, Altstätten, Switzerland), HyFlex EDM (Coltène/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.