Purpose: In order to evaluate the positional uncertainty of internal organs during radiation therapy for treatment of liver cancer, we measured differences in inter- and intra-fractional variation of the tumor position and tidal amplitude using 4-dimentional computed radiograph (DCT) images and gated orthogonal setup kilovolt (KV) images taken on every treatment using the on board imaging (OBI) and real time position management (RPM) system. Materials and Methods: Twenty consecutive patients who underwent 3-dimensional (3D) conformal radiation therapy for treatment of liver cancer participated in this study. All patients received a 4DCT simulation with an RT16 scanner and an RPM system. Lipiodol, which was updated near the target volume after transarterial chemoembolization or diaphragm was chosen as a surrogate for the evaluation of the position difference of internal organs. Two reference orthogonal (anterior and lateral) digital reconstructed radiograph (DRR) images were generated using CT image sets of 0% and 50% into the respiratory phases. The maximum tidal amplitude of the surrogate was measured from 3D conformal treatment planning. After setting the patient up with laser markings on the skin, orthogonal gated setup images at 50% into the respiratory phase were acquired at each treatment session with OBI and registered on reference DRR images by setting each beam center. Online inter-fractional variation was determined with the surrogate. After adjusting the patient setup error, orthogonal setup images at 0% and 50% into the respiratory phases were obtained and tidal amplitude of the surrogate was measured. Measured tidal amplitude was compared with data from 4DCT. For evaluation of intra-fractional variation, an orthogonal gated setup image at 50% into the respiratory phase was promptly acquired after treatment and compared with the same image taken just before treatment. In addition, a statistical analysis for the quantitative evaluation was performed. Results: Medians of inter-fractional variation for twenty patients were 0.00 cm (range, -0.50 to 0.90 cm), 0.00 cm (range, -2.40 to 1.60 cm), and 0.00 cm (range, -1.10 to 0.50 cm) in the X (transaxial), Y (superior-inferior), and Z (anterior-posterior) directions, respectively. Significant inter-fractional variations over 0.5 cm were observed in four patients. Min addition, the median tidal amplitude differences between 4DCTs and the gated orthogonal setup images were -0.05 cm (range, -0.83 to 0.60 cm), -0.15 cm (range, -2.58 to 1.18 cm), and -0.02 cm (range, -1.37 to 0.59 cm) in the X, Y, and Z directions, respectively. Large differences of over 1 cm were detected in 3 patients in the Y direction, while differences of more than 0.5 but less than 1 cm were observed in 5 patients in Y and Z directions. Median intra-fractional variation was 0.00 cm (range, -0.30 to 0.40 cm), -0.03 cm (range, -1.14 to 0.50 cm), 0.05 cm (range, -0.30 to 0.50 cm) in the X, Y, and Z directions, respectively. Significant intra-fractional variation of over 1 cm was observed in 2 patients in Y direction. Conclusion: Gated setup images provided a clear image quality for the detection of organ motion without a motion artifact. Significant intra- and inter-fractional variation and tidal amplitude differences between 4DCT and gated setup images were detected in some patients during the radiation treatment period, and therefore, should be considered when setting up the target margin. Monitoring of positional uncertainty and its adaptive feedback system can enhance the accuracy of treatments.