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The purpose of the present study was to evaluate the frequency of dentinal microcracks observed after root canal preparation with XP-endo Shaper (XP), Reciproc Blue (RB), and ProTaper Universal (PTU) instruments using micro-computed tomographic (μCT) analysis.Thirty extracted mandibular first and second molars with mesial roots having 2 separate canals with an angle between 10°and 20° were randomly assigned to 3 experimental groups (n = 10) according to the different nickel-titanium systems used for root canal preparation: XP, RB, and PTU. The specimens were scanned using μCT imaging before and after root canal preparation. Then, preoperative and postoperative cross-sectional images of the teeth were evaluated to identify the presence of dentinal defects. For each group, the number of microcracks was determined as a percentage rate. The McNemar test was used to determine significant differences before and after instrumentation. The level of significance was set at P ≤ .05.No new dentinal microcracks were observed in the XP and RB groups. The PTU system significantly increased the percentage rate of microcracks compared with preoperative specimens (P < .05).Root canal preparations with XP and RB systems might not induce the formation of new dentinal microcracks on the mesial roots of mandibular molars. Further assessments are suggested for the examination of the morphology of microcracks after the use of these instruments.