HER2 overexpression and amplification have been studied as a therapeutic and prognostic target in a number of human cancers, including esophageal, gastric, and colorectal adenocarcinomas. However, HER2 status has not been well investigated in primary small intestinal adenocarcinoma, probably because of its rarity. In this study, we conducted immunohistochemical analysis and fluorescence in situ hybridization (FISH) for HER2 on 49 primary nonampullar small intestinal adenocarcinomas. The results showed a complete lack of HER2 protein expression in 47 cases (96%) by immunohistochemical analysis. Only 2 cases (4%) showed a 1+ staining pattern. No tumors exhibited 2+ or 3+ HER2 immunoreactivity. By FISH, none of the tumors, including those with 1+ HER2 immunoreactivity, exhibited HER2 gene amplification. These observations demonstrate that HER2 protein overexpression and gene amplification are infrequent events, if they occur at all, in small intestinal adenocarcinoma. Thus, routine immunohistochemical and/or FISH testing for HER2 for potential targeted anti-HER2 therapy may not be beneficial for patients with primary small intestinal adenocarcinoma.