Background: Ductoscopic resolution is insufficient to distinguish the flat lesions and microcalcifications that indicate malignancy in patients with pathologic nipple discharge (PND). This study evaluated the feasibility of imaging intraductal epithelial layers, premalignant, or malignant lesions using catheter-integrated optical coherence tomography (OCT) through the nipple orifices. Methods: Mastectomy specimens were prospectively obtained from patients who had undergone simple or skin-sparing mastectomies for either malignancy (n = 14) or prophylaxis (n = 4). Ductoscopy was used to ensure that the OCT catheter was safely inserted through the nipple orifice of the ex vivo specimens. Cross-sectional OCT scanning of the ductal epithelial layers was performed to acquire high-resolution images of approximately one million pixels. The abnormal lesions identified on the OCT images were evaluated using correlating histopathologic analyses. Results: Fourteen out of 18 mastectomy specimens could be cannulated and distended using ductoscopic instruments and saline. Only 4 out of 18 specimens had presented with clinical PND. To confirm that the ductoscopic trocars were located inside the ducts, 14 specimens were randomly selected and explored with ductoscopy. The OCT catheter was able to insert through the ductoscopic trocars in 10 specimens and revealed one in situ ductal cancer (DCIS) in a cancer-involved specimen and one florid ductal hyperplasia in a prophylactic specimen. Both lesions were confirmed by histopathological correlations. The OCT scanning did not detect malignant lesions in 80% of the specimens without PND; however, the OCT scanning did detect microcalcifications and undefined lesions. Conclusion: This is the first published study to confirm the feasibility of intraductal breast OCT to show millimetric Florid Ductal Hyperplasia and a DCIS in a prophylactic and cancer involved specimen, respectively.