PURPOSE: To prospectively determine if estrogen receptor (ER)-negative human epidermal growth factor receptor type 2 (HER2)-positive and ER-negative HER2-negative breast cancers have distinguishing clinical and imaging features with use of retrospectively identified patients and tissue samples. MATERIALS AND METHODS: This HIPAA-compliant study was institutional review board approved. Informed consent was obtained from living patients and waived for deceased patients. Mean patient age at diagnosis was 53 years (range, 31-84 years). Clinical history; histopathologic, mammographic, and breast sonographic findings; and HER2 status as determined with immunohistochemistry or fluorescent in situ hybridization were evaluated in 56 women with ER-negative breast cancer. Imaging appearances and clinicopathologic characteristics were correlated with tumor HER2 status. P < .05 indicated a significant difference. RESULTS: Lesion margins on mammograms (P = .028) and sonograms (P = .023), calcifications on mammograms (P = .003), and clinical cancer stage at diagnosis (P = .029) were significantly associated with HER2 status. In contrast to ER-negative HER2-negative tumors, ER-negative HER2-positive tumors were more likely to have spiculated margins (56% vs 15%), be associated with calcifications (65% vs 21%), and be detected at a higher cancer stage (74% vs 57%). CONCLUSION: Biologic diversity of cancers may manifest in imaging characteristics, and, conversely, studying the range of imaging features of cancers may help refine current molecular phenotypes. (c) RSNA, 2008.