Objective This study aimed to compare the differences among four kinds of mechanical Ni-Ti files including T-Flex, Reciproc Blue (RB), ProTaper Gold (PTG), and ProTaper Universal (PTU) in dentinal microcrack generation after root canal preparation in vitro by using micro-computed tomography (micro-CT) analysis. Methods A total of 32 freshly extracted double-root-canal premolars with an angle not exceeding 10° were selected and established as root canal preparation models in vitro. Then, the specimens were randomly assigned to four experimental groups (n=8) according to the different Ni-Ti systems used for root canal preparation: group T-Flex, group RB, group PTG, and group PTU. The voxel size of the micro-CT was set at 17.18 μm. Pre- and post-operative cross-sectional images of roots (n=56 940) were scanned and analyzed to identify the presence of dentinal microcracks. The results of each group were expressed by the quantity and percentage of sectional images with microcracks. McNemar test was used to determine whether a significant difference existed in the existence of dentinal microcracks before and after instrumentation. The level of significance was set at P<0.05. Results Overall, 11.04% of the images presented dentinal defects (n=6 288). Dentinal microcracks were observed in 9.82%, 10.79%, 12.27%, and 11.25% of the post-instrumentation images from groups T-Flex, RB, PTG, and PTU, respectively. However, all these dentinal microcracks were already present in the corresponding pre-operative images. No new microcrack of premolars were generated after the root canal preparation utilizing the aforementioned systems. Conclusion Denti-nal microcracks already existed in advance in extracted teeth before root canal preparation. Root canal preparation using the T-Flex, RB, PTG, and PTU systems did not induce the formation of new dentinal microcracks on the straight root canals of premolars.