Objective: To evaluate the prevalence of dentin defects, including partial and complete cracks and fractures, after root canal preparation in molars with Reciproc and WaveOne reciprocating instruments. Material and Methods: Fifty mandibular first and second molars with mature apices were selected to endodontic in vitro instrumentation. Ten teeth were unprepared and served as the control, and the remaining forty teeth were divided into two groups, being twenty corresponding to each reciprocating system. Reciproc and WaveOne systems were used in a reciprocating working motion, under the same conditions, to prepare the two mesial canals. Roots were then sectioned 2, 4 and 6 mm from the apex, and the cut surface was observed under a microscope using 20-fold magnification and checked for the presence of fractures and incomplete cracks. Results: none of the evaluated groups presented fractures, and the control group showed no incomplete cracks. Overall evaluation showed statistical differences between these two groups and the control group (p=0.017) for the prevalence of incomplete cracks, but no significant differences were obtained between Reciproc and WaveOne groups (p>0.05). Reciproc group presented more incomplete cracks on 4-mm sections compared with the control group on the same section (p=0.0326). Conclusion: Root canal preparation with both reciprocating instruments resulted in incomplete cracks, but not fractures. At the level of 4 mm from the apex of the canals, the Reciproc system produced significantly more incomplete cracks. Considering both systems, WaveOne utilization resulted in lesser structural alterations on dentin considering the middle portion of the roots.