Background: Intraoperative specimen radiography is used by surgeons to evaluate partial mastectomy specimens to ensure that the lesion in question has been adequately removed. We sought to determine whether three-dimensional (3D) specimen imaging would better predict margin status and reduce the need for re-excision than conventional two-dimensional (2D) imaging. Methods: A prospective study using standard 2D as well as 3D specimen imaging was undertaken. Surgeons were asked whether the additional orthogonal view would change their management (i.e., result in further margins being taken intraoperatively), and the impact of this on final margin status. Results: Of the 100 women participating in the study, pathology results were available in 99. Of these, 10 had no residual tumor in the definitive specimen (either due to neoadjuvant chemotherapy, or due to the entire tumor being removed in the core biopsy). The remaining 89 patients formed the cohort of interest. 21 (23.6%) had DCIS, 18 (20.2%) had invasive cancer, and 50 (56.2%) had both. The median tumor size of the largest component was 1.7 cm (range; 0.2 – 8.1 cm). Based on the conventional two-dimensional imaging, surgeons stated they would take more tissue in 26 patients (29.2%). Of the 63 patients in whom no further excision would have been indicated on 2D imaging, the 3D imaging changed management in 4 patients (6.3%). Two of these patients would have had positive margins if the intraoperative resection done on the basis of the 3D imaging would have been omitted. Patients who surgeons felt, either by 2D or 3D intraoperative imaging, warranted intraoperative re-excision tended to have a closer initial margin than those in whom re-excision was thought not to be needed on the basis of intraoperative imaging (median 1.0 vs. 2.0 mm, p=0.038). Furthermore, patients in whom an immediate intraoperative margin was taken (either due to imaging or as a matter of routine) were less likely to require subsequent re-excision (13.4% vs. 40.9%, p=0.012). Conclusions: While 3D specimen imaging changes management in only 6.3% of cases, these data highlight the role of intraoperative immediate re-excision in potentially reducing re-excision rates.