To evaluate the diagnostic performance for margin assessment of specimen radiography (SR) in breast conserving surgery (BCS) using radioactive I 125 -seed localization (RSL). Methods: The clinical, radiographic and histopathological data of women who underwent BCS after pre-operative RSL with intraoperative SR during nine consecutive years were analyzed. The histological margin and radiographic margin outcomes on SR were compared and results of intraoperative re-excisions were investigated. Results: A consecutive series of 448 women with invasive carcinoma (n = 211), ductal carcinoma in situ (DCIS) (n = 79) and a combination of DCIS and invasive carcinoma (n = 158) were included. The median minimal margins for the radiological masses and microcalcifications measured on SR were 14 mm and 11 mm, respectively. Based on a radiological cut-off SR margin value of 1 mm, the overall sensitivity, specificity, and positive and negative predictive values (PPV and NPV) were 21.0%, 95.0%, 26.0%, and 94.0%, respectively. The area under the receiver-operating curve was 0.73. Intraoperative re-excisions based on SR were performed in 31 (6.9%) patients; histopathological examination of the additional excised tissue revealed DCIS or invasive carcinoma in 6 (19.4%) patients. Hence, SR was beneficial for 6/448 patients (1.3%), and unnecessary intraoperative re-excisions were performed in 20/448 patients (4.5%). The number need to treat is 75; this implies that per 75-SR one resection with involved margins is prevented. Conclusion: SR has a moderate diagnostic performance for margin involvement using RSL. A more accurate intraoperative margin assessment tool is warranted.