Aim: The aim of the present study was to investigate the incidence of cracks in root dentin after root canal preparation with ProTaper Next, HyFlex CM and Hyflex EDM rotary file systems. Methods: 60 single rooted mandibular premolars were selected. All the specimens were divided into four groups of 15 teeth each. Group I– unprepared root canal serve as control. Group II– were prepared with ProTaper Next to size 25/0.06, Group III– were prepared with Hyflex CM to size 25/0.04 and Group IV– were prepared with Hyflex EDM to size 25/–. After root canal preparation, all the roots were horizontally sectioned at 2mm and 4mm from the apex, and the sections were then observed under Confocal Laser Scanning Microscope with 4X magnification. The absence/presence of cracks was recorded, and the data were analyzed with a chi–square test. The significance level was set at P = .05. Results: No cracks were observed in the control group. Vertical root fractures were not observed in any of the groups. The Hyflex EDM file system caused fewer cracks (10%) than the ProTaper Next (20%) and Hyflex CM (16.7) file systems. However, there were no significant differences in crack formation among different file systems (P > .05). Conclusions: Within the limitations of this in vitro study, all of the instrumentation systems used in this study created cracks in the root dentin. The Hyflex EDM file system tended to cause fewer dentinal cracks compared with the ProTaper Next and Hyflex CM file systems.