Background Recent advances in molecular targeted therapy have identified HER2 as an important target for anti-cancer therapy in gastric cancer (GC). Although the clinical relevance and prognostic significance of HER2 in breast cancer has been well acknowledged, it remains controversial in GC. Methods HER2 expression was investigated in two independent series of GC by immunohistochemical staining. One series corresponded to 1,414 cases of whole-tissue sections and the other corresponded to 595 cases of tissue microarrays (TMAs). Results were compared and correlated with clinicopathologic parameters. Results HER2-positivity was detected in 12.3% of whole-tissue sections and 17% of TMAs. Among samples scored 3+, 90.1% stained ≥50% of the tumor area, but only 40.9% in score 2+ cases stained ≥50% of the tumor area. In whole-tissue sections, HER2-positivity was correlated with age ( P  = 0.002), histological type (differentiated or intestinal, P  < 0.001), lymphovascular invasion ( P  = 0.005), and lymph node metastasis ( P  = 0.009). In TMAs, HER2-positivity was correlated only with age ( P  = 0.003) and histological type ( P  < 0.001). Multivariate analyses of the differentiated GC subgroup revealed that HER2-positivity was an independent poor prognostic factor ( P  = 0.042). The cases with HER2-positive in ≥50% of the tumor area showed worse prognosis than those of <50% ( P  = 0.021). Conclusions Despite discrepancies in the results from whole-tissue sections and TMAs, HER2 overexpression was positively correlated with aggressive biological behavior and was an independent poor prognostic factor for recurrence in differentiated GCs. Therefore, HER2-positive GCs should be considered for adjuvant trastuzumab therapy.