Background: Prevention of dentinal Crack formation is of utmost importance in endodontic treatment. This study aimed to evaluate the effect of RaCe, ProTaper, and V-Taper rotary systems on dentinal Crack formation in three Root regions during endodontic treatment. Methods: Seventy human mandibular first molars were selected randomly, and their distal Roots were used. Ten samples were assigned to the control group, and sixty samples were assigned to three groups (n = 20). Each group was prepared with RaCe, ProTaper, or V-Taper rotary files according to the manufacturers’,instructions. A stereomicroscope was used to view Cracks at ×40 magnification. Friedman’, s, chi-squared, and Kruskal-Wallis tests were used for statistical analysis of data (P < 0. 05). Results: A comparison of the three rotary systems did not reveal any significant differences in the number of Cracks between the three Root thirds evaluated (P > 0. 05). A comparison of the number of Cracks in the coronal, middle, and apical thirds in each rotary system showed that the number of Cracks in the middle third was fewer than that in the two other thirds only in the V-Taper group (P < 0. 05), with no significant differences in the ProTaper and RaCe groups between the different Root regions (P > 0. 05). Conclusion: The application of RaCe, ProTaper, and V-Taper rotary files resulted in a similar rate of Crack formation in endodontic treatment. V-Taper files created the minimum number of Cracks in the middle third.