ProTaper Next (Dentsply Maillefer, Ballaigues, Switzerland) (PTN) and One Shape New Generation (MicroMéga, Besançon, France) (OSNG) belong to a relatively new generation of rotary nickel-titanium (NiTi) files. Scientists keep improving features of endodontic files in order to obtain anatomically shaped and cleaned root canals and avoid canal transportation, for a better outcome of the endodontic treatment. For the current study, the aim was to evaluate and assess the changes in root canal morphology after instrumentation with PTN and OSNG by using micro-computed tomography (CT). This high-tech resolution tomography allows a much more detailed analysis of the root canal anatomy and its transformation after rotary instrumentation. We have selected 10 mandibular molars; before and after canal preparation, the samples were distributed in two homogeneous groups (PTN and OSNG groups) and submitted to standardized radiographs and micro-CT (SkyScan1172, Kontich, Belgium). From the three-dimensional (3D) images obtained from the scanning, we were able to perform a two-dimensional (2D) (perimeter, area and roundness), respectively a 3D (volume, surface area, structure model index) analysis, before and after root canal instrumentation. Results did not revealed important statistical differences among the two groups in relation to the curvature and volume of the root canals before instrumentation; after rotary instrumentation, there was a substantially increase of volume and surface area for all the samples (p<0.05). The two types of instruments preserved the original canal path, maintaining a continuous, safe and adequate shape and taper of the root canals.