Introduction: Root canal instrumentation achieves chemical and mechanical preparation before obturation. However instrumentation may result in dentinal defects, decreasing the strength of endodontically treated teeth. Objectives: This study was designed to evaluate dentinal defects formed after root canal instrumentation by two single file systems using reciprocating and rotation motion versus manual instrumentation. Methodology: This study was conducted on 45 mesial roots of mandibular first molars. Teeth were divided into three groups of 15 specimens each. Pre-instrumentation cone beam computed tomography images were taken to identify defects possibly present in the specimens, to have an objective comparison with post-instrumentation images. Root canals were instrumented as follows; Group (1): reciprocating primary Wave One instrument, Group (2): rotary One Shape instrument, Group (3): manual instrumentation with K-Flexofiles. Post-instrumentation cone beam computed tomography images were used to assess dentinal defects formed. Results: The three groups showed dentinal defects formation after instrumentation, with the highest number of defects found in group (2) (One Shape group), however no statistically significant difference was found between the three groups. Conclusions: Dentinal defects formed as a result of instrumentation weren’t affected by the instrumentation technique used.