BACKGROUND: Image-guided spinal instrumentation reduces the incidence of implant misplacement. OBJECTIVE: To assess the accuracy of intraoperative computed tomography (iCT)-based neuronavigation (iCT-N). METHODS: In 35 patients (age range, 18-87 years), a total of 248 pedicle screws were placed in the cervical (C1-C7) and upper and midthoracic (T1-T8) spine. An automated iCT registration sequence was used for multisegmental instrumentation, with the reference frame fixed to either a Mayfield head clamp and/or the most distal spinous process within the instrumentation. Pediculation was performed with navigated drill guides or Jamshidi cannulas. The angular deviation between navigated tool trajectory and final implant positions (evaluated on postinstrumentation iCT or postoperative CT scans) was calculated to assess the accuracy of iCT-N. Final screw positions were also graded according to established classification systems. Mean follow-up was 16.7 months. RESULTS: Clinically significant screw misplacement or iCT-N failure mandating conversion to conventional technique did not occur. A total of 71.4% of patients self-rated their outcome as excellent or good at 12 months; 99.3% of cervical screws were compliant with Neo classification grades 0 and 1 (grade 2, 0.7%), and neurovascular injury did not occur. In addition, 97.8% of thoracic pedicle screws were assigned grades I to III of the Heary classification, with 2.2% grade IV placement. Accuracy of iCT-N progressively deteriorated with increasing distance from the spinal reference clamp but allowed safe instrumentation of up to 10 segments. CONCLUSION: Image-guided spinal instrumentation using iCT-N with automated referencing allows safe, highly accurate multilevel instrumentation of the cervical and upper and midthoracic spine. In addition, iCT-N significantly reduces the need for reregistration in multilevel surgery.