Introduction: The aim of this study was to determine the incidence of crack initiation and propagation in apical root dentin after retreatment procedures performed by using 2 rotary retreatment systems and hand files with additional instrumentation. Methods: Eighty extracted mandibular premolars with single canals were selected. One millimeter from the apex of each tooth was ground perpendicular to the long axis of the tooth, and the apical surface was polished. Twenty teeth served as the control group, and no preparation was performed. The remaining 60 teeth were prepared to size 35 with rotary files and filled with gutta-percha and AH Plus sealer. Specimens were then divided into 3 groups (n = 20), and retreatment procedures were performed with the following devices and techniques: Pro Taper Universal retreatment files, Mtwo retreatment files, and hand files. After retreatment, the additional instrumentation was performed by using size 40 Pro Taper, Mtwo, and hand files. Digital images of the apical root surface were recorded before preparation, after instrumentation, after filling, after retreatment, and after additional instrumentation. The images were then inspected for the presence of any new apical cracks and propagation. Data were analyzed with the logistic regression and Fisher exact tests. Results: All experimental groups caused crack initiation and propagation after use of retreatment instruments. The Pro Taper and Mtwo retreatment groups caused greater crack initiation and propagation than the hand instrument group (P < .05) after retreatment. Additional instrumentation with Pro Taper and Mtwo instruments after the use of retreatment instruments caused crack initiation and propagation, whereas hand files caused neither crack initiation nor propagation (P < .05). Conclusions: This study showed that retreatment procedures and additional instrumentation after the use of retreatment files may cause crack initiation and propagation in apical dentin.