Purpose: To evaluate the prostate patient setup and intra‐fraction organ motion error distributions by analyzing initial setup shift and target tracking data obtained from the Calypso system. Method and Materials: The spatial coordinates of the centroid of three implanted markers implanted in the prostates of 13 patients were monitored by our Calypso system with 10Hz sampling frequency. Patients were initially setup to tattoos and then shifted to the Calypso localized locations. Measurements were performed for average 32 fractions of average time about 8 minutes each. For each patient, the systematic setup correction and standard‐deviation (SD) over all fractions was obtained for the left‐right (LR), anterior‐posterior (AP) and superior‐inferior (SI) axes. Systematic setup error was obtained as the SD of the systematic setup errors of all patients. Similarly, the systematic and random components of prostate intra‐fractional motion were obtained from the tracking data. For all patients, correlation coefficients were calculated among the three axes of tracking data; the cumulative probability of total 3D displacement from initial setup was also calculated. Results: The systematic and random components of the initial setup errors for LR, AP and SI axes are 2.4mm, 2.6mm and 3.2mm, respectively and 3.6mm, 2.7mm and 3.3mm. The corresponding systematic and random components of prostate intra‐fractional motion errors are 0.3mm, 0.5mm and 0.6mm, and 0.6mm, 1.1mm and 1.0mm, respectively. The correlation between AP and SI motion was significant. The 3D displacement data indicates that 11 of 13 patients have 94.4% or higher probability of prostate motion within 5mm from the Calypso setup positions. However, one patient showed 28.7% probability of motion beyond 5mm. Conclusion: In our patient population, residual systematic and random error due to intra‐fraction prostate motion is small, once inter‐fraction setup‐organ motion errors have been eliminated by Calypso‐guided online setup. Supported in part by grant P01 CA 116162.