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Introduction: The aim of this study was to evaluate the influence of rotary (ProTaper Next [PTN; Dentsply Maillefer, Ballaigues, Switzerland] and ProTaper Gold [PTG, Dentsply Maillefer]) and reciprocating (WaveOne Gold [WOG, Dentsply Maillefer]) systems in dentinal microcrack generation after the preparation of curved root canals using micro-computed tomographic analysis., Methods: Twenty-four human mandibular molars with curved roots were scanned in a micro-computed tomographic device using an isotropic resolution of 6.78 μm and randomly assigned into 1 of 3 experimental groups (n = 8) according to the root canal instrumentation system used (PTN, PTG, or WOG). Then, the root canals were prepared up to PTN X2, PTG F2, and WOG Primary instruments in the PTN, PTG, and WOG groups, respectively. After canal preparation, each specimen was scanned again. Pre- and postoperative cross-sectional images of the roots (N = 35,304) were analyzed to identify the presence of dentinal microcracks., Results: Overall, 26% of the images presented dentinal defects (n = 9188). Dentinal microcracks were observed in 24.6%, 26%, and 27.4% of the postinstrumentation images from the PTN, PTG, and WOG groups, respectively. However, all of these dentinal microcracks were already present in the corresponding preoperative images. No new microcracks were generated after the preparation of curved root canals of mandibular molars using the aforementioned systems., Conclusions: Root canal instrumentation with PTN, PTG, and WOG systems did not induce the formation of new dentinal microcracks., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)