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Introduction: Success in endodontics depends on canal preparation, disinfection, and obturation. Cleaning and shaping play a pivotal role in successful endodontic therapy. An important factor in preventing procedural mishaps and further complications is the quantity of dentine removed during instrumentation because the fracture resistance of the root is directly influenced by the amount of dentine that remains after instrumentation. Aim: To evaluate and compare the remaining dentine thickness of the root canal after instrumentation with three different rotary file systems using Cone Beam Computed Tomography (CBCT) imaging. Materials and Methods: Thirty permanent extracted, single-rooted teeth were collected. The samples were decoronated at the level of the Cementoenamel Junction (CEJ). A pre-instrumentation CBCT scan was taken. Access opening and working length were determined. The samples were randomly assigned to three groups: Group I - Edge Endo X7, Group II- Hero Gold, Group III - Neo-endo flex files. Biomechanical preparation was carried out using the respective files. To evaluate the remaining dentine thickness, a comparison was made with the pre-instrumentation CBCT scan. Statistical analysis was performed using One-way ANOVA. Results: Upon inter-group comparison of the pre-instrumentation group, no significant difference (p≥0.05) was observed. Post-instrumentation, it was noted that the remaining dentine thickness in Group I (p≤0.05) at the apical third was significantly less than in Group II (p=0.007) and Group III (p=0.042). Maximum preservation of dentine was seen with Hero gold files, whereas the minimum preservation of dentine was seen with Edge Endo X7. Conclusion: Hero gold files exhibited less removal of dentine apically, whereas Edge Endo X7 showed more removal of dentine apically.