Statement of the Problem: Vertical root fractures are catastrophic events that often result in tooth extraction. Many contributing factor are associated with increasing incidence of vertical root fracture. Root canal preparation is one of the predisposing factors which can increase the root susceptibility to vertical fracture.Purpose: The aim of this study was to compare the effects of three different instrumentation techniques on vertical root fracture resistance of endodontically treated teeth.Materials and Methods: In this study, 120 freshly extracted mandibular premolar teeth of similar dimensions were decoronated and randomly divided into control (n=30), nickel-titanium hand K-file (HF, n=30), BioRaCe rotary file (BR, n=30), and WaveOne reciprocating single-file (WO, n=30) groups. After cleaning and shaping the root canals, AH26 was used as canal sealer, and obturation was completed using the continuous wave technique. The root canals were embedded vertically in standardised autopolymerising acrylic resin blocks, and subjected to a vertical load to cause vertical root fracture. The forces required to induce fractures were measured using a universal testing machine. ANOVA and Tukey’s post-hoc test were used to analyse the data.Results: All experimental groups showed statistically significant reductions in fracture resistance as compared with the control group. There was a statistically significant difference between the HF and BR groups. The WO group did not differ significantly from the HF group or the BR group.Conclusion: All three instrumentation techniques caused weakening of the structure of the roots, and rendered them susceptible to fracture under lesser load than unprepared roots. The fracture resistance of roots prepared with the single-file reciprocating technique was similar to that of those prepared with NiTi hand and rotary instrumentation techniques.