Abstract Aim: This study analyzed the two-dimensional (2D) and three-dimensional (3D) parameters and canal transportation using micro-CT in roots prepared with three instrumentation systems preceded or not by glide path. Materials and Methods: Sixty mesial canals of mandibular molars with severe curvature angles (≥25°) were divided randomly (n = 10): G1-Twisted-file-adaptive (TFA); G2-Reciproc (RC); G3-One-Shape (OS); G4-ProGlider (PG)+TFA; G5-PG+RC; and G6-PG+OS. Area, perimeter, circularity, and major and minor diameters at the apical 5 mm and volume and surface area and canal transportation were evaluated. Data were analyzed using two-way analysis and Tukey's test (α = 0.05). Results: RC showed significantly higher areas and lower perimeters and diameters than TFA (P < 0.001). Increases in root canal volumes promoted by RC and OS were similar (P = 0.979). There was no statistically significant difference in canal transportation (P = 0.083). RC generally promoted major changes in the 2D and 3D parameters. Conclusions: The RC system caused a significantly higher perimeter and area increase than the TFA system. The preceding use of PG in the instrumentation techniques did not influence the evaluated parameters.