Objective: The current study's goal was to evaluate the incidence of dentinal micro-cracks in root canal dentin following root canal preparation with ProTaper Next (Dentsply Maillefer, Ballaigues, Switzerland), TruNatomy (Dentsply Maillefer, Ballaigues, Switzerland), and Race Evo (FKG Dentaire SA, La Chaux-de- Fonds, Switzerland) rotary systems. Methods: A total of 60 single rooted maxillary premolars with single canal were selected. Teeth were decoronated and roots lengths were standardized to 14mm. Roots were then randomly allocated into four groups (n=15) according to root canal preparation system that had been used; one negative control group that hadn't been prepared and three experimental groups; ProTaper Next group, Race Evo group and TruNatomy group. At 3, 6, and 9 mm from the apex, all of the roots were sectioned perpendicular to the long axis after root canal preparation, and each section was examined under a stereomicroscope at 25 X magnification (Leica microsystems Ltd, Germany). Results: The negative control group did not show any cracks. At 3mm ,6mm and 9mm root levels ProTaper Next showed the highest prevalence of micro-cracks followed by Race Evo then TruNatomy with a statistically significant difference between systems (p-value =0.006,0.042,0.018) respectively. Conclusion: TruNatomy and Race Evo rotary systems showed lower incidence of dentinal microcracks in comparison to ProTaper Next rotary system.