The intraductal spread of breast cancer is a major cause of local recurrence following breast-conserving therapy. To properly understand this pathology, three-dimensional (3D) cancer localization within the mammary ductal-lobular system (MDLS) is necessary. To this end we generated computer-assisted 3D reconstructions of all MDLSs using 2-mm-thick serial sections of surgically resected specimens. We then analyzed the characteristics of intraductal spread of breast cancer. In our study of quadrantectomy specimens from patients with primary invasive breast carcinoma, the intraductal spread of breast cancer was found to be continuous from the invasive tumor and spreading along the mammary glandular tree. The pattern is categorized into three types: the central type, the peripheral type, and the extensive type. The central type was found to be most common. A 3D analysis of total mastectomy specimen from a patient with primary non-invasive breast carcinoma revealed regional intraductal spread extending within and filling a single MDLS. The analysis also revealed the presence of ductal anastomoses connecting adjacent MDLSs. These ductal anastomoses were found to be an anatomical risk factor for extensive intraductal spread of breast cancer across multiple MDLSs. To minimize residual non-invasive components of breast carcinoma in the conserved breast, which is strongly associated with the outcome of local control of breast-conserving therapy, it is necessary to determine the optimum surgical margins in a flexible, patient-specific manner. This determination should be based on anatomical characteristics of the MDLS, such as those identified in the present study.