Objective To investigate the effects of root canal preparation treated by endodontists of different levels in different difficulty associated with the root canal anatomy factors. Methods Totally 422 teeth with 901 root canals were randomly selected from the Conservative Dentistry Department, West China Stomatology Hospital, and these teeth were respectively treated by junior specialist postgraduates, senior specialist postgraduates and endodontic specialists. Grading criteria of root canal risk factor and root canal therapy difficulty assessment were found based on 6 items: Tooth type, working length, root curvature, calcification, number of canals, and the previous endodontic treatment. The effects of root canal preparation with different difficulty in different groups were analyzed with R×C Chi-square test. Results Success rates of root canal preparation in junior group and senior group during three difficulty groups were statistically different(Pgroup Ⅱ> group Ⅲ. The operator factors in root canal therapy difficulty group Ⅰ and group Ⅱ were not significantly different (P>0.05). But in root canal therapy difficulty group Ⅲ, the endodontic specialist group have highest success rate in root canal preparation. Conclusion The cases of root canal treatment should be treated by endodontist of corresponding level according to the difficulty. In difficult cases, endodontic specialist can provide better root canal preparation result.