Introduction: The aim of the present study was to evaluate the frequency of dentinal microcracks observed after root canal preparation with ProTaper Universal (PTU; Dentsply Tulsa Dental Specialties, Tulsa, OK), ProTaper Gold (PTG; Dentsply Tulsa Dental Specialties), Self Adjusting File (SAF; ReDent Nova, Ra'anana, Israel), and XP-endo Shaper (XP; FKG Dentaire, La Chaux-de-Fonds, Switzerland) instruments using micro computed tomographic (CT) analysis. Methods: Forty extracted human mandibular premolars having single-canal and straight root were randomly assigned to 4 experimental groups (n = 10) according to the different nickel titanium systems used for root canal preparation: PTU, PTG, SAF, and XP. In the SAF and XP groups, the canals were first prepared with a K-file until #25 at the working length, and then the SAF or XP files were used. The specimens were scanned using high-resolution micro computed tomographic 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. 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. Results: The PTU system significantly increased the percentage rate of microcracks compared with preoperative specimens (P < .05). No new dentinal microcracks were observed in the PTG, SAF, or XP groups. Conclusions: Root canal preparations with the PTG, SAF, and XP systems did not induce the formation of new dentinal microcracks on straight root canals of mandibular premolars.