Abstract Background: In preparation for adjuvant breast radiation therapy (RT), several small but permanent skin tattoo marks are often placed to assist with surface alignment for treatment setup and delivery. With advancements in surface-guided technologies, it may be possible to achieve setup accuracy with a “tattoo-less” approach, which can improve cosmetic and quality of life outcomes. This study evaluates the feasibility, accuracy, and potential dosimetric implications of a tattoo-less setup technique. Method: A retrospective analysis of 10 patients diagnosed with right-sided breast cancer who received adjuvant whole breast RT using a tattoo-less approach were included in this study. All patients received an initial whole breast dose of 42.56 Gray (Gy) in 16 daily fractions using a tangential three-dimensional conformal planning technique. This was followed by a 10Gy boost to the lumpectomy site delivered in 4 daily fractions. Daily setup for whole breast treatment was performed by aligning each patient to their individual reference breast surface generated from the planning computed tomography (CT) images via the Vision RT system. Bony landmarks were then verified via daily kV imaging, and translational shifts were recorded for each fraction. The kV imaging was performed to confirm the treatment positioning since the tattoo-less approach was new to the department. For 5 patients, dosimetric differences between initial surface alignment and shifts performed based on bony landmarks were evaluated by recalculating the original clinical plan with a new isocenter position using kV image shifts for each of their 16 treatment fractions. A plan sum of the 16 recalculated plans for each patient was used in further dosimetric analysis. Boost fractions were not included in this study, as setup was verified using surgical clips instead of bony anatomy. Results: Translational shifts for 158 fractions were reviewed as no kV imaging was performed for the other 2 fractions. The mean absolute kV imaging shift was 0.29 cm (CI (95%): 0.25, 0.32; range: 0 to 1.25) in the vertical axis, 0.28 cm (0.23, 0.32; 0 to 1.27) in the longitudinal axis, and 0.24 cm (0.20, 0.28; 0 to 1.98) in the lateral axis. Clinical plans for 5 patients were evaluated with a mean absolute kV shift of 0.27 cm (range: 0.15 to 0.39) in the vertical, 0.32 cm (0.13 to 0.77) in the longitudinal, and 0.29 cm (0.18 to 0.38) in the lateral directions. Dosimetric analysis revealed a slight increase in mean heart dose from the original clinical plan, with an average difference of 4.12 cGy (range: 0.7 to 10.5 cGy). The maximum increase in V20Gy of the ipsilateral lung was 2.7%. Less than 1% of change was observed in the maximum plan dose, as well as in the V95% coverage, expect for one patient, whose V95% was decreased from 99.6% to 94.6%. This difference was caused by a large longitudinal shift which could be attributed to possible patient movement. Conclusions: These findings demonstrate the feasibility and accuracy of a tattoo-less treatment approach in a small cohort of breast cancer patients receiving whole breast RT. Further investigation with larger cohorts is warranted. Citation Format: Xinxin Zhang, Swati Mamidanna, Yin Zhang, Xiao Wang, Ning Yue, Shicha Kumar, Lindsay Potdevin, Maria Kowzun, Mridula George, Lara Hathout, Bruce Haffty, Nisha Ohri. Evaluation of a surface-guided and tattoo-less approach to setup for whole breast radiotherapy [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 PO5-22-02.