The 2018 American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) update modified the interpretation guidelines for human epidermal growth factor receptor 2 (HER2) testing by incorporating immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results in a subset of cases. Importantly, the new guidelines eliminate "equivocal" results, as well as the use of alternative chromosome 17 probes as the primary strategy for resolving the indeterminate FISH results. Herein, we investigate the predicted impact of implementing the 2018 ASCO/CAP guidelines on HER2 assessment by FISH in breast cancers, using data from a single institution. We compared the HER2 status of 1542 consecutive cases of breast carcinoma, interpreted by 2013 and 2018 ASCO/CAP guidelines. In total, 10.7% (165/1542) of the cases had a different final interpretation by 2018 guidelines compared with 2013 guidelines, including 70 previously HER2-positive cases reclassified as negative, four previously negative cases reclassified as positive, and 91 previously equivocal cases reclassified as negative. Overall, the number of HER2-positive cancers was reduced by 66 cases (4.3% reduction in the HER2 positivity rate). The newly HER2-negative cases were mostly estrogen receptor positive (90%), progesterone receptor positive (80%), stage 1 (60.9%), and grade 1-2 (59.4%) cancers; 70% of them had been designated as HER2 positive only after the use of an alternative chromosome 17 FISH probe after an intially equivocal result from the standard CEP17 probe. Overall, implementing the revised 2018 HER2 guidelines is predicted to change the HER2 results of 10.7% of breast cancers, mainly by reclassifying previously equivocal to negative results.