Purpose: Rotation greater than one degree can severely compromise the accuracy of radiotherapy treatments. Prostate intrafractional rotation is not negligible because peristaltic motion has a time scale that is shorter than the delivery time of a single fraction. The objectives of this study were (a) to investigate a clinically feasible method to calculate tumor rotation with radiopaque markers; (b) to evaluate the accuracy of the method and (c) to quantify prostate intrafractional rotation in intensity‐modulated arc radiotherapy (IMAT). Methods: 2D kilovoltage (kV) cone‐beam computed tomography (CBCT) images were acquired for prostate cancer patients with three implanted gold fiducial markers as surrogates for the tumor position during fractionated IMAT. Post‐treatment, 3D marker trajectories were determined from the 2D projection images by maximum likelihood estimation of a 3D probability density function. Rotations about each axis (roll (right/left), pitch (superior/inferior) and yaw (anterior/posterior)) were then calculated based on the iterative closest point algorithm (ICP) method, with the origin set at the centroid of the three markers. Results: Intrafractional rotation was studied for10 patients, 268 fractions. Average roll, pitch and yaw were calculated as 0.85±1.16, 0.41±0.51 and 0.37±0.4 degrees, respectively. Roll was the predominant rotation among the three axes which is consistent with literature. Rotations showed a similar pattern among different fractions for the same patient. The pattern acquired at the first few fractions could be used as a prediction for the rest of the fractions. Conclusion: This study demonstrated a novel method to quantify intrafractional tumor rotation from 2D kV projections with radiopaque markers. Intrafractional rotation is small but significant for certain cases. Real‐time radiopaque marker‐based adaptive radiotherapy with rotation correction could make a positive impact on reducing treatment margins. This project is supported by an NHMRC Australia Fellowship and NHMRC project grant 1034060.