Objectives: The present in-vitro study was undertaken to evaluate and compare fracture strength of tooth roots following canal preparation by hand and rotary instrumentation and, further, to determine the effect of these different canal preparation techniques on the direction and pattern of fracture lines. Study design: 60 freshly extracted human mandibular premolars were randomly divided into four groups of 15 teeth each. In Group I, canals were prepared with step-back technique using standardized nickel-titanium K-files. In Groups II, III and IV canals were prepared with crown-down technique using Quantec LX, ProTaper and V-Taper rotary files respectively. After obturation, a vertical load was applied by means of a spreader inserted into the canal until fracture occurred. The roots were subsequently examined under a dissecting microscope with 20X magnification to determine the direction and pattern of fracture lines. The results were statistically analyzed using Kruskal-Wallis and Mann-Whitney U-test. Results: The Mean fracture load was 10.1 ± 3.9 kg for K-files, 12.9 ± 5.2 kg for Quantec LX files, 13.6 ± 5.1 for ProTaper files and 14.3 ± 6.1 kg for V-Taper files, but the differences between four groups were not statistically significant (p>0.05). Most of the fracture lines observed were in bucco-lingual direction, followed by proximal and compound fractures. Conclusion: Increased canal taper as advocated by Quantec LX, ProTaper and V-Taper files did not increase the fracture susceptibility of mandibular premolars any more than conventional step-back K-file preparations and may even increase the fracture resistance.