Introduction: File fracture is one of the main procedural mishaps in endodontic treatment. The aim of this in vitro study was to compare the fracture rate of three NiTi rotary systems; Hero 642, Mtwo and FlexMaster in artificial canals. Methods and Materials: In this study, bovine long bone was used. After primary preparation of bones, longitudinal sections with 4-cm diameter were cut and encoded. Subsequently, semicircular sections were prepared. A total number of 500 canals were created in the same way; the upper 3 mm of the canals were initially prepared with orifice shapers and then canals were filed with FlexMaster files sizes 25/0.02 and 25/0.04 to 13 mm of canal length. The prepared canals were assigned into 3 groups of the following systems: Hero 642, Mtwo and FlexMaster. Six selected instruments were used from each system; the files were applied 13 mm along the canals for 10 sec with manufacturer’s suggested speed and torque. The number of the canals prepared by each file before its separation was recorded; finally the data was analyzed with ANOVA test. Results: Mean number of prepared canals in Mtwo, FlexMaster and Hero groups before file separation was 15, 25 and 32, respectively. Conclusion: Results of this study showed that the number of prepared canals by Hero 642 was more than FlexMaster and Mtwo systems.