The objective of this study is to evaluate the setup discrepancy between Brain Lab ExacTrac (kv) and Orthogonal Novalis Tx (kv) imaging systems and to obtain an optimal CTV-PTV margin. We recruited 35 consecutive brain tumor patients, immobilized with non-invasive thermoplastic BrainLab mask, between August 2016 and August 2017. The position verification done by ExacTrac, Infrared Positioning System was followed by a set of images by OBI at 0° and 90° position. The images were autofused and compared with DRR generated by TPS. The extent of translational and rotational shift was then applied and documented. The translational inter-fractional setup error (mean ± SD) in X-axis detected by OBI and ET was 0.002 ± 0.227 and −0.042 ± 0.084, respectively. In Y-axis, the error detected was −0.01 ± 0.176 and 0.075 ± 0.079, respectively, whereas in Z-axis the error with OBI and ET was 0.329 ± 0.521 and 0.051 ± 0.091, respectively. The P-value of inter-fractional errors in X, Y, and Z axes was 0.294, 0.013, and 0.004, which is statistically significant in Y- and Z-axes. Rotational inter-fractional setup errors (mean ± SD) for OBI and ET were −0.101 ± 0.257 and −0.076 ± 0.102, respectively. The rotational setup errors detected by ET in pitch and roll dimension are −0.003 ± 0.084 and −0.085 ± 0.073, respectively. The data obtained were put in Van Herk’s formula (PTV margin = 2.5 ∑ + 0.74 σ), and CTV-PTV margin was calculated. CTV-PTV margin for ET was 2.70, 2.72, and 2.94 mm and 3.38o, 2.84o, and 2.51o in lateral, longitudinal, vertical and pitch, roll, yaw dimension, respectively. The CTV-PTV margin for OBI was 7.40, 6.20, and 5.30 mm and 7.27o in lateral, longitudinal, vertical, and yaw dimension, respectively. Daily setup verifications that use the Brain LAB Exac Trac 6D image-guided system are very useful for brain tumor treatment; nevertheless, OBI kv imaging can be used for FRT of brain tumor with extended margin.