This study was designed to evaluate usefulness of additional fluorescence in situ hybridization (FISH) using other reference genes on chromosome 17 for assessment of HER2 status in invasive breast cancers with increased centromere 17 copy number, and to compare this approach with conventional methods based on the 2007 and 2013 ASCO/CAP guidelines. We performed FISH with probes for SMS, RARA, and TP53 on 253 breast cancers with centromeric probe CEP17 copy number ≥ 2.6 using tissue microarrays. If one or more gene had a mean copy number <2.6, the largest number for that gene(s) was chosen as an alternative to CEP17 copy number. Of the 243 cases in which re-grading was possible, only 2 had copy numbers ≥ 2.6 for RARA, SMS, and TP53. Of the 151 breast cancers which were considered HER2 non-amplified by the 2007 ASCO/CAP guidelines using the HER2:CEP17 ratio, 42 (27.8%) were re-graded as amplified and 33 (21.8%) as equivocal after FISH using additional reference genes. Of the 101 HER2-non-amplified cases by the 2013 ASCO/CAP guidelines, 2 (2.0%) were reclassified as amplified and 24 (23.8%) as equivocal. Of 46 equivocal cases, 35 (76.1%) were re-graded as amplified. After re-grading, HER2-amplified cases were significantly increased, and the concordance between HER2 FISH and HER2 immunohistochemistry decreased. And some pathologic features of the cases which were designated to have HER2 amplification after additional FISH were not compatible with those of HER2-amplified breast cancers. The use of additional reference genes has been suggested as an option for accurate assessment of HER2 status in breast cancers with increased CEP17 copy number. However, this has limitations in that it can cause over-grading of HER2 status in tumors that lose the new reference genes. Thus, at present, it seems that additional FISH using other reference gene such as SMS, RARA, and TP53 for the cases with increased CEP17 copy number is not suitable for daily practice.