The HER2 status of breast cancers is routinely assessed using the testing algorithm standardized by the American Society of Clinical Oncology and the College of American Pathologists, which includes immunohistochemical analysis of HER2 protein expression and fluorescence in situ hybridization (FISH) analysis of HER2 gene copy number. Studies have suggested that HER2 status may differ between primary and metastatic tumors. Furthermore, studies have suggested that trastuzumab may convert HER2 status from positive in a primary tumor to negative in a metastatic site. Assessment of HER2 status is affected by aspects of the pathologic analysis, including the method of tumor sampling, the method of specimen fixation, whether immunohistochemical or FISH analysis is used. In this review, we discuss the clinical significance of HER2 discordance in breast cancer. Moreover, the importance of tumor sample handling in assessing HER2-positivity.