Background: Image-guided navigation is routinely used in spine surgery to improve placement of pedicle screws. However, most reports have relied on two-dimensional X-ray evaluation to determine accuracy of screw positioning. In this study, computed tomography (CT) and O-arm imaging enabled a detailed three-dimensional comparison of screw placement. The objective was to compare the accuracy of pedicle screw placement with intraoperative X-ray versus O-arm image-guided navigation. Methods: This was a retrospective analysis of image-guided pedicle screw placement in patients who underwent spinal instrumentation. Post-operative CT and O-arm imaging allowed grading of screw accuracy based on pedicle breaches. Clinical outcomes included patient and operative factors. Results: Pedicle screws were placed in 208 patients (1116 screws). Three-dimensional O-arm guidance was utilized for 126 patients, while the remainder underwent two-dimensional X-ray imaging and post-operative CT assessment. O-arm navigation was associated with improved pedicle screw accuracy: pedicle breaches were more likely to be low grade (odds ratio 2.84, p=0.001) and less likely to be medium grade (odds ratio 0.35, p=0.007) or high grade (odds ratio 0.31, p=0.025). Conclusions: This study provided a detailed comparison of surgical accuracy with X-ray versus O-arm guidance. Navigation with O-arm imaging is associated with benefits in spinal instrumentation, without impacting operative risks for patients.