Aim: To evaluate and compare in vitro the dentinal crack formation in root canal dentin after root canal instrumentation with hand K-Flex files, ProTaper Next, and self-adjusting engine-driven files. Materials and methods: Ninety-two human mandibular first molar teeth were randomly divided into four groups (n = 23) as per the instrumentation protocol: group I—unprepared teeth (control); group II—hand K-Flex files (Sybron Endo); group III—ProTaper Next X1 and X2 (Dentsply Maillefer); group IV—self-adjusting file (ReDent Nova, Israel). All the roots were sectioned perpendicular to the long axis of the tooth at 9 mm, 6 mm, and 3 mm from the apex and thereby obtaining sixty-nine samples, which were then subjected to a stereomicroscopic examination for detection of dentinal cracks. Statistical analyses were done using the Chi-square test with SPSS (version 19), and p value was set at p < 0.05. Results: Statistically significant differences were seen between the instrumented groups. No dentinal cracks were found in the unprepared roots and those prepared with hand K-Flex files (0/23). Self-adjusting-file-instrumented group showed significantly less incidence of crack formation when compared to the ProTaper Next group with p = 0.001. Conclusion: Self-adjusting file is an efficient engine-driven NiTi instrument for root canal instrumentation with the least occurrence of crack formation in the root canal dentin compared to the ProTaper Next system. Clinical significance: The introduction of NiTi rotary file systems has metamorphosed the endodontic treatment by their better cutting efficiency and cleaning potentiality. Yet, evidenced-based clinical studies are to be conducted on the incidence of microfractures that can lead to vertical root fractures, which will be produced irrespective of the motion kinematics and design feature and thereby compromising the clinical maintenance of the endodontically treated teeth.