Improved pedicle screw insertion accuracy has been reported with the assistance of computer tomography-based navigation. Studies also indicated that fluoroscopy-based navigation offers high accuracy and is comparable to CT-based assistance. However, different population characteristics and assessment methods resulted in inconsistent conclusions. We searched OVID, Springer, and MEDLINE databases to conduct a meta-analysis of the published literature specifically looking at accuracy of pedicle screw placement with different navigation methods. Subgroups and descriptive statistics were determined based on the subject type (in vivo or cadaveric), navigational method, and spinal level. A total number of 7,533 pedicle screws were summarised in our database with 6,721 screws accurately inserted into the pedicles (89.22%). Overall, the median placement accuracy for the in vivo CT-based navigation subgroup (90.76%) was higher than that with the use of two-dimensional (2D) fluoroscopy-based navigation (85.48%). We concluded that CT-based navigation could provide a higher accuracy in the placement of pedicle screws for all subgroups presented. In the lumbar level, 2D fluoroscopy-based navigation was comparable with CT-based navigation. Discrepancy between the two navigation types increased in the thoracic level for the in vivo populations, where there was less potential in the use of 2D fluoroscopy-based navigation than CT-based navigation.