The purpose of the present study was to study the occurrence of radicular dentinal defects after using EDTA gel during preparation of the root canal with ProTaper Universal (Dentsply Maillefer, Switzerland), HyFlex (ColteneWhaledent, Allstetten, Switzerland), and ProTaper Next (Dentsply Maillefer) rotary instruments. Forty-eight single rooted premolars were selected. Twelve teeth were instrumented with hand files; another 12 teeth were instrumented with the ProTaper Universal system up to size F3 and the remaining 24 teeth were shaped with Hyflex CM 25/0.6 and ProTaper Next X3. EDTA gel was used during the root canal preparations of each tooth. After the completion of root canal instrumentation, the roots were sectioned at 3mm,6mm and 9mm followed by stereomicroscopic observation of the sections. The absence/presence of radicular dentinal defects was documented, and the data analysis was done with a chisquare test. The significance level was set at P = 0.05. The ProTaper Next and HyFlex CM instruments caused fewer cracks (33.33% and 50%) than the ProTaper Universal instrument (66.7%) (P < .05). However, there were no significant variances in radicular dentinal defects formation between the Hyflex CM and ProTaper Next groups (P > 0.05). Within the confines of this in vitro study, all of the instrumentation systems used induced radicular defects in the root dentin. The ProTaper Next and HyFlex instruments tended to cause fewer dentinal cracks compared with the ProTaper Universal instrument.