Background: The intratumoral heterogeneity of HER2 expression in gastric cancer (GC) is a major challenge for identifying patients (pts) who would benefit from anti-HER2 therapy. The aim of this study is to evaluate the significance of re-evaluation of the HER2 status by repeat endoscopic biopsy in pts with HER2-negative GC on initial endoscopic biopsy. Methods: Pts with unresectable or metastatic gastric/gastroesophageal junction (GEJ) adenocarcinoma who would receive 1st line chemotherapy were eligible if the HER2 was negative on the initial endoscopic biopsy. HER2 positivity was defined as IHC 3+ or IHC 2+/FISH+ using the GC scoring system. A repeat endoscopic biopsy was performed in ≥6 different primary tumor sites immediately after obtaining initial HER2-negative results. Results: From May 2011 to April 2013, a total of 183 pts were enrolled. Baseline characteristics at the time of the initial biopsy were as follows: tumor location, GEJ~fundus/body~antrum/diffuse stomach = 22 (12.0%)/115 (62.9%)/46 (25.1%); Lauren classification, intestinal/diffuse/mixed = 53 (29.0%)/111 (60.7%)/19 (10.4%); and HER2 IHC score, 0/1/2 = 149 (81.4%)/26 (14.2%)/8 (4.4%). The median number of biopsy pieces was 5 (range, 1-15) and 10 (1-15) in the initial and repeat biopsy, respectively (p<0.0001). As HER2 positive tumor was identified in 16 pts, HER2 positivity rate on repeat biopsy was 8.7% (95% CI 4.6-12.8%). The detection of HER2 positivity on repeat biopsy was associated with tumor location (diffuse stomach vs others = 0% vs 11.7%, p=0.013), Bormann type (IV vs others = 0% vs 11.7%, p=0.013), and the HER2 IHC score on the initial biopsy (0 vs 1/2 = 6.7% vs 18.2%, p=0.045). In multivariate analysis, the HER2 IHC score (1/2 vs 0, OR = 3.30; p=0.041) was an independent predictor of HER2 positivity in repeat biopsy. Conclusions: In pts with metastatic or unresectable GC, repeat endoscopic biopsy could detect HER2-positive GC which initial biopsy had missed. As anti-HER2 therapy improves the survival of pts with HER2 positive GC, in pts who showed HER2 negativity on initial biopsy, repeat biopsy should be considered subsequently.