This study evaluated the increase of the instrumented area and dentin thickness in the mesial and distal aspects of mesial canals of mandibular molars after the use of Gates Glidden (GG), LA Axxess (LA) and Orifice Shaper (OS) instruments. A total of 53 canals from 27 mandibular molars were embedded in resin and divided into 3 groups. The roots were sectioned 3 mm below the cementoenamel junction and the images were captured before and after instrumentation. The increase of the instrumented area in terms of percentages and the remaining dentin thickness, in mm, at the mesial and furcal aspects were calculated using the Image tools software. Data were analyzed using the Kruskal-Wallis and Dunn's test. The significance level was set at 5%. All instruments promoted cervical flaring with different amounts of dentin removal at the mesial and distal aspects of the root canals. Statistically significant difference (p<0.05) was found between LA and all other instruments after using the first instrument. Regarding dentin thickness, no significant difference (p>0.05) was found between the mesial and distal walls in all groups. It may be concluded that LA 35.06 and GG 3 burs produced the thinnest dentin walls, and thus their use in mesial canals of mandibular molars should be done with caution.