A monoclonal antibody to HER2 protein is widely used in the treatment of patients with HER2-overexpressing breast cancer and has also been found to exhibit antitumor activity in human gastric cancer cells that overexpress HER2. The purpose of this study was to evaluate the frequency of HER2 overexpression and concordance between the results for protein expression and gene amplification in both surgical and biopsy specimens of gastric cancer as assessed with two commercial kits, one for immunohistochemistry (IHC) and the other for fluorescence in situ hybridization (FISH). The specimens consisted of formalin-fixed, paraffin-embedded sections of biopsy specimens and surgically resected tumors from 200 cases of invasive gastric cancer that had been treated surgically at the National Cancer Center Hospital East. The lesions were analyzed with the IHC kit, and expression was graded by the United States Food and Drug Administration (FDA)-approved grading system. Gene amplification was evaluated by FISH. IHC revealed HER2 overexpression in 46 of the 200 (23%) cases. The FISH assay was technically successful in 199 cases (99.5%), and gene amplification was observed in 54 cases (27.1%). The concordance rate between the results obtained by IHC and FISH was 86.9%. The concordance rate between the findings in the surgically resected tumors and the 200 pre-treatment biopsy specimens was 88.7%. HER2 expression can be assessed in gastric cancer with a commercial kit as previously reported in breast cancer. Even small biopsy specimens were found to be suitable for evaluating gastric cancer for HER2 overexpression.