Background:The success of endodontic treatment depends on completely removing all vital and necrotic pulp tissue, microorganisms, and microbial toxins from the root canal. During the cleaning and shaping process, the canal is shaped through the interaction between instruments and the dentin walls. These interactions can create several temporary stress points within the dentin, which may lead to the development of dentinal defects, craze lines, or microcracks. As a result, the techniques employed for shaping root canals carry the risk of starting crack formation, which, under functional pressure, could progress into complete fractures. This study was aimed to compare and evaluate the incidence of dentinal microcrack formation during root canal preparation using ProTaper Universal, ProTaper Gold, NeoEndo Flex and Hyflex EDM files. Materials and Methods: A total of 95 freshly extracted single rooted premolars with single straight canal were selected and stored in normal saline. Each specimen was examined using dental operating microscope to exclude cracked samples. Specimens then decoronated using a diamond disc and water as a coolant to a standardized length of 14 mm. Canal patency was determined by passing 10 K file passively and the working length was established 1 mm short of the file length at which the file become visible at the apical foramen. Canal was then enlarged upto15 k size file. Teeth samples were then divided into 5 groups having 19 teeth each. Group 1: Control Group (Uninstrumented teeth), Group 2 : Protaper Universal, Group 3 : Protaper Gold, Group 4 : NeoEndo Flex ,Group 5 : Hyflex EDM. After completion of cleaning and shaping procedure (to a standard apical diameter of 25), canal was rinsed with distilled water. Root was then horizontally sectioned at 3 mm, 6 mm and 9 mm from the apex with diamond disc at low speed under water cooling. All the sections were evaluated under stereomicroscope for presence of cracks and images were recorded. Cracks were observed as lines extending from the root canal lumen to the dentin or from the outer surface into the dentin. Results: All the file systems used in this study showed dentinal microcracks. ProTaper Universal exhibited the highest percentage of dentinal defects, while Hyflex EDM displayed the lowest among the file systems used (ProTaper Universal, ProTaper Gold, NeoEndo Flex and Hyflex EDM). The defects were present more in the apical third (3 mm) as compared to 6 mm (middle third) and 9 mm (coronal third) sections