Trastuzumab provides a survival benefit in patients with human epidermal growth factor receptor 2 (HER2)-amplified/overexpressed advanced gastric and gastroesophageal junction cancers (GC/GJCs). However, the optimal method for testing is unclear. The aim of this study was to assess interlaboratory agreement on HER2 scoring in GC/GJC to aid the development of a robust testing algorithm for diagnostic practice in Australia. Nine laboratories assessed the HER2 status of 100 GC/GJC tissue samples by immunohistochemistry (IHC) and in situ hybridization (ISH) [chromogenic (CISH) or silver (SISH)] using both HER2 copy number and HER2:chr17 (chromosome 17) ratio. Results were compared with reference fluorescence ISH (FISH). Interlaboratory agreement on IHC3+ scoring was good (?=0.76), and there was good/very good agreement between IHC (positivity defined as IHC3+) and ISH when HER2 copy number was used (?=0.72 to 0.87). Agreement on CISH/SISH scoring was good/very good when HER2 copy number was used (?=0.68 to 0.86), and agreement between CISH/SISH and FISH using HER2 copy number was very good (?=0.88 to 0.91). Agreement was reduced when HER2:chr17 ratio was used. The good agreement for HER2 copy number determined by bright-field ISH suggests that this is the optimal method for testing in GC/GJC cases. An IHC3+ score was strongly predictive of a positive ISH result, although agreement for all IHC scores was only moderate, suggesting that IHC triage before ISH testing would be the most cost-effective strategy. However, because of the unique features of GC/GJC samples and the difficulty of ensuring consistent HER2 staining in the community setting, it is recommended that HER2 status in advanced GC/GJC be determined by both IHC and ISH in the same laboratory.