It is critical to evaluate the overexpression of human epidermal growth factor receptor 2 (HER2) adequately in breast cancer cases. The aim of the present study is to assess the characteristics and usefulness of a novel monoclonal antibody, clone SV2-61gamma. The overexpression of HER2 was studied on paraffin sections of 50 breast cancers using a polyclonal antibody assay (HercepTest) and monoclonal antibodies (clones SV2-61gamma and CB11). Gene amplification of HER2 was determined by fluorescent in situ hybridization (FISH) using a HER2/CEP17DNA probe kit. Fifty tumors were scored as 0 (50.0%), 1+ (26.0%), 2+ (4.0%), or 3+ (20.0%) by immunohistochemistry using a monoclonal antibody, clone SV2-61gamma. The specificities of immunohistochemistry were 72.0% for the polyclonal antibody, 97.0% for SV2-61gamma, and 92.0% for CB11. The concordances with FISH were 78.0% for the polyclonal antibody, 98.0% for SV2-61gamma, and 94.0% for CB11. The positive predictive value of SV2-61gamma (92.0%) was higher than those of the polyclonal antibody (50.0%) and CB11 (79.0%). A monoclonal antibody clone SV2-61gamma showed very specific staining, with the best concordance with FISH, and it is useful for the evaluation of HER2 overexpression.