Background: We recently reported the clinical significance of IHH in trastuzumab (T-mab) based chemotherapy using surgical specimens; namely homogeneously HER2 positive GC was associated with longer survivals compared with heterogeneously HER2 positive GC (ASCO-GI 2015). However, three quarters of patients were diagnosed as having GC by endoscopic biopsy and no data are available as to whether IHH using endoscopic biopsy specimens predict T-mab efficacy. In this study, we addressed clinical usefulness of IHH using endoscopic biopsy specimens. Methods: Patients who received T-mab based chemotherapy and had endoscopic biopsy specimens available for IHC test were retrospectively examined. When all biopsy specimens which contained cancer cells were diagnosed HER2 IHC3+, it was defined as “Inter-specimens homogeneously HER2 positive (Inter-homo)”, and the others were defined “Inter-hetero”. When all tumor cells showed HER2 overexpressed within a specimen, it was defined “Intra-specimen homogeneously HER2 positive (Intra-homo), and the others were defined “Intra-hetero”. Progression-free survival (PFS) and overall survival (OS) were estimated using Kaplan-Meier methods and compared using the log-rank test. Results: 57 patients were enrolled in this study. The median age was 67 years old and 70% were male. PS 0, GEJ cancer, intestinal type histology, and visceral metastasis (lung or liver) were found in 74% , 25% , 60% , and 53% , respectively. After the median follow-up period of 12.1 months, the median PFS and OS were 7.6 and 16.9 months, respectively. Significant survival differences were shown only in the comparison between inter-homo and inter-hetero; inter-homo group (n = 34) showed significantly longer PFS (8.5 vs. 5.1 months, HR 0.52 95%CI 0.27-0.98, p = 0.038) and OS (27.6 vs. 12.1 months, HR 0.27 95%CI 0.12-0.61, p = 0.001), respectively. Conclusions: Our data suggest that, in contrast to surgical specimens, the assessment of HER2 heterogeneity using endoscopic biopsy specimens may be useful for negative selection of patients who are unlikely to benefit from T-mab based chemotherapy.