This study investigated the efficacy of three newly introduced rotary endodontic systems: Genius ProFlex, TruNatomy, and ZenFlex. Forty-five mandibular molars with root canal curvatures <5° were utilized. Micro-CT scans were performed pre- and post-preparation to assess apical transportation, centralization, dentin thickness, and canal volume alterations. Eight instruments of each diameter underwent cyclic fatigue testing. Dentin thickness on mesial and distal walls showed no significant differences among ZenFlex, TruNatomy, and Genius ProFlex at 1, 2, 3, and 4 mm from the apical foramen and root canal orifice (p>0.05). Centering ability varied in the mesiolingual canal (p<0.05). No notable differences were observed in transportation (p>0.05). Genius ProFlex demonstrated lower volumetric changes (p<0.05). There were significant differences in cyclic fatigue, with higher values for Genius ProFlex and lower values for TruNatomy (p<0.05). The three NiTi rotary instruments are safe and efficient for root canal preparation, with Genius ProFlex exhibiting superior cyclic fatigue resistance.