AbstractPurposeTo assess dynamic tumor tracking (DTT) target localization uncertainty for in‐vivo marker‐based stereotactic ablative radiotherapy (SABR) treatments of the liver using electronic‐portal‐imaging‐device (EPID) images. The Planning Target Volume (PTV) margin contribution for DTT is estimated.MethodsPhantom and patient EPID images were acquired during non‐coplanar 3DCRT‐DTT delivered on a Vero4DRT linac. A chain‐code algorithm was applied to detect Multileaf Collimator (MLC)‐defined radiation field edges. Gold‐seed markers were detected using a connected neighbor algorithm. For each EPID image, the absolute differences between the measured center‐of‐mass (COM) of the markers relative to the aperture‐center (Tracking Error, (ET)) was reported in pan, tilt, and 2D‐vector directions at the isocenter‐plane.Phantom studyAn acrylic cube phantom implanted with gold‐seed markers was irradiated with non‐coplanar 3DCRT‐DTT beams and EPID images collected.Patient Study: Eight liver SABR patients were treated with non‐coplanar 3DCRT‐DTT beams. All patients had three to four implanted gold‐markers. In‐vivo EPID images were analyzed.ResultsPhantom Study: On the 125 EPID images collected, 100% of the markers were identified. The average ± SD of ETwere 0.24 ± 0.21, 0.47 ± 0.38, and 0.58 ± 0.37 mm in pan, tilt and 2D directions, respectively.Patient Study: Of the 1430 EPID patient images acquired, 78% had detectable markers. Over all patients, the average ± SD of ETwas 0.33 ± 0.41 mm in pan, 0.63 ± 0.75 mm in tilt and 0.77 ± 0.80 mm in 2D directions The random 2D‐error, σ, for all patients was 0.79 mm and the systematic 2D‐error, Σ, was 0.20 mm. Using the Van Herk margin formula 1.1 mm planning target margin can represent the marker based DTT uncertainty.ConclusionsMarker‐based DTT uncertainty can be evaluated in‐vivo on a field‐by‐field basis using EPID images. This information can contribute to PTV margin calculations for DTT.