To our knowledge, there are no universally accepted, evidence-based guidelines for how to resolve the HER2 status of tumors demonstrating equivocal amplification. The present study was based on 17 breast core biopsy specimens demonstrating invasive carcinoma with equivocal HER2 amplification, defined as an HER2/chromosome 17 centromere ratio of 1.8 to 2.2. Each case had a corresponding resection specimen, on which HER2 immunohistochemical and repeated fluorescence in situ hybridization analyses were performed. A definitive change in HER2 status based on the resection specimen occurred in 10 (59%) of 17 cases, with 4 patients (24%) becoming eligible for trastuzumab therapy and 6 (35%) triaged as ineligible. These results suggest that genetic and protein expression heterogeneity exists in tumors that show low-level HER2 gene copy numbers. For the purposes of uniform clinical management, HER2 status should be evaluated on a larger tumor sample if the core biopsy specimen demonstrates an equivocal result. These results support the recent American Society of Clinical Oncology/College of American Pathologists recommendations for further testing in cases with equivocal HER2 results.