Objectives: The aim of this study was to compare in vitro root fracture resistance following root canal filling with AH 26 using lateral condensation, BeeFill, and Thermafil techniques. Study Design: Eighty extracted human mandibular premolars with similar dimensions were selected. In order to standardize the roots, measurements were taken in two separate regions of the teeth?at the cemento-enamel junction and 8 mm apically from the junction?buccolingual as well as mesiodistal for every tooth. Teeth were then randomly divided into five groups (n=16). With the exception of the non-prepared group (Group 1), instrumentation was done in all groups. In group 2, instrumentation but no filling was performed; in group 3, the obturation was done with AH 26 + gutta-percha; in group 4, with AH 26 + BeeFill and in group 5, AH 26 + a Thermafil obturator was used. All the roots were mounted vertically in copper rings and filled with acrylic resin, exposing 8 mm of the coronal part. A universal testing machine was used for the strength test. Results: The results were analyzed using the one-way ANOVA test. The significance between the groups was tested with Temhane?s T2 test. The results indicate that instrumentation of root canals had a significant effect on fracture resistance (p<0.05). In addition, there were no differences between the root canal obturation techniques; furthermore, these techniques did not create a statistically important resistance to vertical fracture (p>0.05). Conclusions: The results suggest that instrumentation of root canals significantly weakens the tooth structure to fracture and the root canal obturation techniques that are used are not able to form reinforcement.