Objectives: The aim of this in vitro study was to evaluate and compare the incidence of dentinal defects caused by different nickel titanium rotary systems like ProTaper, WaveOne and OneShape files. Methodology: In this study, 150 extracted human mandibular second premolar teeth were selected and decoronated using diamond disk with water cooling, leaving roots approximately 16 mm from apex. Teeth which showed cracks were excluded. Root canal was prepared with K files, ProTaper files, Wave One files and One Shape files. The root canals which not prepared at all were taken as Control group. Then teeth were sectioned horizontally at 3 mm, 6 mm and 9 mm from the apex. To evaluate cracks, cut cross sections were visualized under Stereo Microscope and images were taken with camera attached with microscope. The chi-square test was performed to compare the appearance of cracked roots between the experimental groups. Results and Conclusion: No defects were found in the unprepared teeth and teeth prepared with hand files showed very less defects. Teeth prepared with ProTaper, WaveOne and OneShape resulted in dentinal defects in percentage 57.78%, 23.33% and 35.56% respectively. No complete crack was found in any sample. More defects were seen in coronal and middle third group. Very fewer percentages of cracks were seen in apical third of all the groups