Introduction: Inapropriate mechanical treatment of root canals can result in unfavourable outcome of root canal treatment. Objectives: The aim of this study was to compare rotational and reciprocal instrumentation techniques by measuring the degree of dentin removal and preserving the original root canal anatomy. Materials and Methods: Twenty permanent mandibular molars extracted for periodontal reasons were mechanically cleansed and randomly divided into two groups of ten teeth each. The first group was instrumented with a rotational instrumentation technique, the F360 system (Komet Dental, Lemgo, Germany), and the second group was instrumented with the Endo-Eze Genius reciprocating system (Ultradent Products, Utah, USA) according to the manufacturer's protocols. Measurements of the canal diameter and canal curvature straightening were obtained using standardised X-ray images taken before and after instrumentation at four levels of the root canal by utilizing computer software Adobe Photoshop CC 2020 (Adobe Systems Incorporated, San Jose, California, USA). Results: Rotational technique resulted in a statistically significant increase in root canal diameter after instrumentation at all levels (all p<0.05). The reciprocating technique significantly increased the canal diameter at all levels (all p<0.05) except at the second level (p=0.063). Regarding the maintenance of root canal anatomy, both techniques lead to canal alignment. When compared to reciprocal technique, rotational instruments caused significantly greater straightening of the curve at the third level (p=0.017). Conclusions: Both instrumentation systems have similar degree of dentine removal, however rotational technique is more prone to cause root canal straigtening.