Objective: The purpose of this study was to evaluate the impact of glide path preparation on the incidence of dentinal defects after preparation of severely curved canals using ProTaper Next, F6 SkyTaper, and One Shape instruments. Method and Materials: One hundred and forty human mandibular molars with mesiobuccal canals with angles of curvatures ranging between 25 and 35 degrees and radii ranging between 4 and 9 mm were assigned to seven homogenous groups (n = 20 teeth per group). In half of the teeth of each of the seven groups a manual glide path (size 15) was created prior to instrumentation. The root canals were instrumented using the three different types of instruments to an apical size 25. One group served as control as no canal preparation was performed. Roots were sectioned horizontally at 3, 6, and 9 mm from the apex and evaluated under a microscope using 25-fold magnification. The presence of dentinal defects (complete/incomplete cracks) was noted and analyzed using Fisher's exact test. Results: No defects were observed in the controls. All canal preparations created dentinal defects. More incomplete and complete cracks were found in the apical (n = 4) and the middle (n = 3) canal third than in the coronal (n = 1), without any significant differences between the three instruments (P > .05). Prior glide path preparation had no significant impact on the incidence of dentinal defects (P > .05). Conclusion: A glide path preparation seems not to be mandatory to reduce the risk of dentinal defects formation when using the tested instruments in severely curved canals. Keywords: dentinal defects, F6 SkyTaper, One Shape, ProTaper Next, single-file systems