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INTRODUCTION: Smoking during breast radiotherapy (RT) may be associated with radiation-induced skin injury (RISI). We aimed to determine if a urinary biomarker of tobacco smoke exposure is associated with increased rates of RISI during and after breast RT. PATIENTS AND METHODS: Women with Stage 0-IIIA breast cancer treated with breast-conserving surgery or mastectomy followed by RT to the breast or chest wall with or without regional nodal irradiation were prospectively enrolled on a multicenter study assessing acute/late RISI. 980 patients with urinary cotinine (UCot) measurements (baseline and end-RT) were categorized into three groups. Acute and late RISI was assessed using the ONS Acute Skin Reaction scale and the LENT-SOMA Criteria. RESULTS: Late Grade 2+ and Grade 3+ RISI occurred in 18.2% and 1.9% of patients, respectively-primarily fibrosis, pain, edema, and hyperpigmentation. Grade 2+ late RISI was associated with UCot group (P= 006). Multivariable analysis identified UCot-based light smoker/secondhand smoke exposure (HR 1.79, P= .10) and smoking (HR 1.60, p = .06) as non-significantly associated with an increased risk of late RISI. Hypofractionated breast RT was associated with decreased risk of late RISI (HR 0.51, P=.03). UCot was not associated with acute RISI, multivariable analysis identified race, obesity, RT site/fractionation, and bra size to be associated with acute RISI. CONCLUSIONS: Tobacco exposure during breast RT may be associated with an increased risk of late RISI without an effect on acute toxicity. Smoking cessation should be encouraged prior to radiotherapy to minimize these and other ill effects of smoking.