Introduction: The purpose of this study was to evaluate the incidence and mode of WaveOne (Dentsply Tulsa Dental Specialties, Tulsa, OK) instrument defects after single use at different endodontic clinics. Methods: A total of 438 WaveOne instruments were collected after clinical use from the 4 specialist clinics over a 12 month period and from 1 graduate program over a 20-month period. The incidence and type of instrument defects were analyzed. The lateral surfaces of part of the defective instruments and fracture surfaces of fractured files were examined using scanning electron microscopy. Unused and clinically used files were examined by a nanoindentation test. Results: Of the 438 WaveOne instruments collected, 42 (9.6%) had defects: 40 (9.1%) were distorted and 2 (0.5%) files had fractured, 1 Small and 1 Primary file. Clear differences in the frequency of defects were found among the 3 file sizes; the occurrence of distortion and fracture were highest with the Small file (21.2% and 0.7%, respectively) followed by the Primary file (4.4% and 0.4%, respectively) (P < .05). No defects were detected on the Large file. The cause of the 2 fractures was shear stress. Instruments from various clinics showed no significantly different occurrence of instrument deformation. Unwinding occurred at 1.2-3.1 mm from the tip. No significant difference in nanohardness was detected among unused and used instruments. Conclusions: The risk of used fracture is very low when files are singly used by endodontists and residents. Unwinding of the files occurred most frequently in the Small file. The frequency of defects of WaveOne instruments were not influenced by the operator.