The purpose of this study was to evaluate the presence of dentinal defects after root canal preparation with hand instruments and two different reciprocating instruments. Sixty freshly extracted mandibular incisor teeth were selected for this in vitro study. On the basis of root length, mesiodistal and buccolingual dimensions, the teeth were allocated into three identical experimental groups (n = 15) and one control group (n = 15). The teeth in the control group were left unprepared. The other groups were: stainless steel hand instruments, WaveOne (R) Primary instruments and RECIPROC (R) R25 instruments. The reciprocating instruments were used with a reciprocating gentle in-and-out motion in a torque-limited electric motor at the appropriate preset mode. Horizontal sections were made 3, 6 and 9 mm from the apex. Samples were stained with methylene blue and viewed through a stereomicroscope. The presence of dentinal defects (fractures, incomplete cracks and craze lines) and their locations were investigated by two endodontists. These data were analysed statistically by Fisher's exact and chi-square tests. No defects were observed in the unprepared group. All instruments caused dentinal defects, with no significant differences between the instrument systems. All experimental groups demonstrated significantly more defects at the 3-mm level in comparison with the unprepared group (p = 0.032). At the other levels, there was no significant difference between the experimental groups and the control group. The use of hand or reciprocating instruments could induce the formation of dentinal defects during root canal preparation.