Objectives: The aim of this study was to compare the formation of dentinal crack after root canal preparation performed with different heat treated single-file systems. Materials and Methods: Forty mandibular premolar teeth were randomly divided into four groups according to the file system to be used during the preparation (n = 10): Group 1: Reciproc Blue; group 2: One Curve; group 3: HyFlex EDM; group 4: (control group) hand files. During the cleaning and shaping, irrigation was performed with a total of 10 ml of 5.25% sodium hypochlorite and 2 mL of distilled water. Then sections were taken from the roots (3, 6 and 9 mm). Sections were examined on a stereomicroscope to determine the presence of cracks. Chi-square test was used for data analysis. Results: There was a significant difference between the groups in dentinal crack frequency (P <.05). HyFlex EDM (13.3%) caused less dentinal crack than One Curve (26.7%) and Reciproc Blue (30%). No cracks were observed in the control group. When 3, 6, and 9 mm were evaluated within themselves, dentinal crack formation did not differ according to the groups (P > 0.05). A statistically higher mean value was obtained in the control group compared to the other groups in terms of working time (P < 0.001). Conclusions: All the files used during root canal preparation formed dentin crack. Hy-Flex EDM caused less dentinal cracks than other file systems. The flexibility of nickel-titanium instruments because of heat treatment seems to have a significant influence on dentinal crack formation.