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Root canal morphology changes during canal preparation, and these changes may vary depending on the technique used. Such changes have been studied in vitro by measuring cross-sections of canals before and after preparation. This current study used nondestructive high-resolution scanning tomography to assess changes in the canals' paths after preparation. A microcomputed tomography scanner (cubic resolution 34 μm) was used to analyze 18 canals in 6 extracted maxillary molars. Canals were scanned before and after preparation using either K-Files, Lightspeed, or ProFile .04 rotary instruments. A special mounting device enabled precise repositioning and scanning of the specimens after preparation. Differences in surface area (ΔA in mm2) and volume (ΔV in mm3) of each canal before and after preparation were calculated using custom-made software. ΔV ranged from 0.64 to 2.86, with a mean of 1.61 ± 0.7, whereas ΔA varied from 0.72 to 9.66, with a mean of 4.16 ± 2.63. Mean ΔV and ΔA for the K-File, ProFile, and Lightspeed groups were 1.28 ± 0.57 and 2.58 ± 1.83; 1.79 ± 0.66 and 4.86 ± 2.53; and 1.81 ± 0.57 and 5.31 ± 2.98, respectively. Canal anatomy and the effects of preparation were further analyzed using the Structure Model Index and the Transportation of Centers of Mass. Under the conditions of this study variations in canal geometry before preparation had more influence on the changes during preparation than the techniques themselves. Consequently studies comparing the effects of root canal instruments on canal anatomy should also consider details of the preoperative canal geometry.