Abstract Objectives To quantify the intra-fraction tumor bed motion using image guidance and fiducial markers to determine optimal PTV margins in early-stage breast cancer patients receiving adaptive radiation therapy SPBI after partial mastectomy. Methods A retrospective cohort of five patients with pT1a-pT2N0, grade 1-2, ER+/PR+ early-stage invasive ductal carcinoma of either the right breast or left breast underwent partial mastectomy and adjuvant five-fraction SPBI using cone beam based online adaptation. A total of 62 CBCTs and 22 total fractions were analyzed after post-processing with an average time span of 39.1±8.9 minutes. Two methods were utilized to determine optimal CTV to PTV margins: one contouring-based and another fiducial-based. Our contouring-based method involved independently contouring on each fraction’s CBCTs including pre-treatment CBCT, intra-treatment CBCT, and post-treatment CBCT. After image registration with the original simulation CT image, these contours were propagated to and combined to create a new union CTV. Using the original CTV on the simulation CT, optimal PTV margin was determined to achieve 95% overlap of the union CTV created from fractionated CBCTs. In addition to the contouring-based method, our fiducial-based method utilized an in-house script to calculate the centroid of all fiducials per CBCT. The average and maximum anterior-posterior, medial-lateral, superior-inferior shifts of fiducials between intra-fraction CBCTs were recorded. Results Using our contouring-based method, we calculated and determined that a uniform 3.5 mm expansion around the original CT simulation CTV is needed to achieve 95% PTV overlap encompassing the pre-treatment, intra-treatment, and post-treatment union CTV. Furthermore, with our fiducial-based method, we calculated average centroid anterior-posterior, medial-lateral and superior-inferior shifts to be the following: 1.51±0.79mm, 1.04±0.55 mm, 1.78±0.94 mm respectively. Average maximum shifts were determined to be 2.13±1.18mm anterior-posterior, 1.45±0.76mm medial-lateral and 2.47±1.40mm superior-inferior shifts. Conclusion With our contouring-based analysis, this preliminarily study suggests CTV to PTV margin of 3.5mm is adequate. On the other hand, our fiducial-based method suggests decreased movement and smaller asymmetric margin. Further CBCT adaptation images will be analyzed. In conjunction with these two methods, we currently recommend utilizing a 3.5mm uniform margin for intrafraction motion and to assess adequate coverage with image guidance during daily adaptation for patients with early-stage breast cancer receiving SPBI. Citation Format: Meng-Lun Hsieh, Justin Visak, Sean Domal, Gabrielle Gard, Steven Montalvo, Liyuan Chen, Xinran Zhong, Zohaib Iqbal, Tingliang Zhuang, Bin Cai, Heejung Kim, Mona Arbab, Prasanna Alluri, Nathan Kim, Mu-Han Lin, David Parsons, Asal Rahimi. Optimal intra-fraction PTV margins for patients receiving stereotactic partial breast radiation (SPBI) using cone beam adaptive radiation therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-22-02.