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INTRODUCTION This study aimed to use micro-computed tomography to analyze the quality of the endodontic preparation of mesial canals in mandibular molars, provided by three instrumentation protocols. METHODS Forty-five extracted mandibular molars with 2 independent mesial canals were selected and initial micro-computed tomography was performed. The initial volume values of the canals were submitted to statistical analysis for paired division. The groups were determined according to the final enlargement of the canal and the work length adopted: G25.06 /+1 mm; G35.05/foramen and G50.01/-1 mm. At the end of each instrumentation sequence, the root canals were scanned and analyzed with regard to the increase in total and apical volume, centralization, and preparation transportation, and the percentage of total and apical uninstrumented walls. RESULTS For the intragroup comparison, the Wilcoxon test was used, and for the intergroup analysis, the Kruskal Wallis and Dunn tests were used, (p<0.05). In the analysis of canal total volume, statistical difference was found between G25.06/+1 mm and the remaining groups (P<0.05). In the apical third, statistical difference was observed between G25.06/+1 mm and G50.01/-1 mm (P<0.05). No statistical difference was found between the groups in terms of centralization and transportation of the preparation or in terms of the percentage of total or apical uninstrumented walls. CONCLUSIONS The preparation of mesial canals of mandibular molars up to larger tip files, but with lower taper at 1mm before the foramen resulted in larger volume of apical preparation, kept the preparation centralized, provided safe apical dentin wear, without excessive cervical wear.