Objective: The aim of this study was to evaluate root canal preparation of two nickel-titanium (NiTi) rotary systems (BioRaCe, FKG, Switzerland and AlphaKite, Komet, Germany) using micro-computed tomography (Micro-CT). Material and Methods: A total of 20 maxillary first molars, extracted for periodontal and prosthetic reasons, were divided into two groups (n = 10) and scanned by SkyScan 1172. Root canals were irrigated with 2.5% NaOCl between each file change, the root canal preparation was performed up to instrument 35.04 of both BioRaCe and AlphaKite rotary systems. After root canal preparation, teeth were rescanned by micro-CT. The amount of canal transportation, centering ability of rotary instruments and canal variations were analyzed. Results: There was no significant difference in canal transportation between the two rotary systems at any level (p > 0.05). Conclusion: BioRaCe and AlphaKite instruments showed minimal canal transportation and good centering ability at all areas of the root canal, with no significant difference between the two systems. Root canal curvature did not seem to influence the results.