The aim of this study was to assess and compare dentinal defect formation caused by three different nickel–titanium (NiTi) rotary file systems in permanent mandibular premolars. Materials and methods: Total of 45 extracted mandibular premolars were selected for present In-vitro study. Hyflex EDM rotary files, ProTaper Next, and Waldent Walflex were used to instrument the root canals. With a slow-speed saw and water cooling, all roots were horizontally sectioned at 3, 6, and 9 mm from the apex. To assess whether a crack existed or not, the sections were examined with a stereomicroscope at a magnification of x 25. The result showing P value ≤ 0.05 was considered statistically significant. Results: There was a significant higher incidence of microcracks at the 6 mm and 9 mm levels of roots in the all gropus (p < 0.05). The HyFlex EDM files produced less dentinal defect when compared with the other groups (P < 0.05) at 3, 6 and 9 mm. Conclusion: Within the limitation of this in vitro study, it can be concluded that nickel-titanium instruments may cause cracks on the root surface. Hyflex EDM tends to produce less number of cracks as compared to Protaper Next and Waldent Walflex.