Introduction: Root canal preparation is a crucial step in the success of endodontic treatment because of the debridement of debris, elimination of bacteria, and facilitating final obturation. Dentinal micro-cracks could be in a horizontal direction or vertical directions at different root levels. The present study aimed to evaluate dentinal micro crack formation after instrumentation with Pro Taper Next, Wave One Gold (Gold-wire) and Trunatomy (DENTSPLY) file systems under the stereomicroscope. Materials and Methodology: Forty single rooted teeth free from any defect were selected and divided into four groups (Control, PTN, WOG and Trunatomy). Experimental groups were instrumented with file system used in the study, and the control group were left unprepared. Samples were further decoronated and sectioned at 3 mm, 6 mm, and 9 mm from the apex perpendicular to the long axis and examined under a stereomicroscope at ×25 for different types of micro-cracks (no fracture, complete fracture, partial fracture, and other fracture). Results: The tables that were demonstrated above denoted the microscopical observation of cracks in the root dentin area after instrumentation. At the coronal level, higher number of cracks was observed in group – 2 that is pro taper next When microscopically observing the cracks at the middle portion, it has been observed that group – 2 showed higher cracks and in the apical portion, higher number of cracks were observed in group – 2. Conclusion: To conclude, it has been established from the study that all the Ni-Ti rotary files are capable of inducing the cracks during the root canal instrumentation. The maximum percentage of cracks was seemed to be noticed in the apical region.