Background: To analyze the feasibility and accuracy of micro–computed tomography (micro-CT) for surgical margin assessment in breast excision specimen. Materials and methods: Two data sets of 30 micro-CT scans were retrospectively evaluated for positive resection margins by four observers in two phases, using pathology as a gold standard. Results of phase 1 were evaluated to define micro-CT evaluation guidelines for phase 2. Interobserver agreement was also assessed (kappa). In addition, a prospective study was conducted in which 40 micro-CT scans were directly acquired, reconstructed, and evaluated for positive resection margins by one observer. A suspect positive resection margin on micro-CT was annotated onto the specimen with ink, enabling local validation by pathology. Main outcome measures were accuracy, sensitivity, specificity, and positive predictive value (PPV). Results: Average accuracy, sensitivity, specificity, and PPV for the four observers were 63%, 38%, 70%, and 22%, respectively, in phase 1 and 72%, 40%, 78%, and 26%, respectively, in phase 2. The interobserver agreement was fair [kappa (range), 0.31 (0.12-0.80) in phase 1 and 0.23 (0-0.43) in phase 2]. In the prospective study 70% of the surgical resection margins were correctly evaluated. Ten specimens were annotated for positive resection margins, which correlated with three positive and three close (<1 mm) margins on pathology. Sensitivity, specificity, and PPV were 38%, 78%, and 30%, respectively. Conclusions: Micro-CT imaging of breast excision specimen has moderate accuracy and considerable interobserver variation for analysis of surgical resection margins. Especially sensitivity and PPV need to be improved before micro-CT–based margin assessment can be introduced in clinical practice.