The aim of this study was to evaluate the dentinal microcrack formation of ProTaper Universal, ProTaper Next, and WaveOne. Sixty extracted mandibular molars were selected. The mesial roots were resected and randomly divided into four groups (n=15). The canals were prepared with hand files (group 1), ProTaper Universal (group 2), ProTaper Next (group 3), and WaveOne (group 4) instrument systems. The roots were separated horizontally at 3, 6, and 9mm from the apex. Digital images were captured at x40 magnification using scanning electron microscopy to detect microcrack formation. Statistical analysis was performed by Pearson Chi-square test. The prevalence of microcracks in group 2, group 3, and group 4 were significantly higher when compared to group 1 (p<0.001). Group 2, group 3, and group 4 demonstrated similar prevalence of microcracks without significant difference (p>0.05) in all sections. All instruments caused microcracks except for hand file. The highest percentage of microcrack was recorded in 3mm section for all groups. SCANNING 37:49-53, 2015. (c) 2014 Wiley Periodicals, Inc.