Clinical benefit from trastuzumab and other anti-human epidermal growth factor receptor-2 (HER2) therapies in patients with HER2-positive gastric cancer (GC) remains limited by primary or acquired resistance. We aimed to investigate the impact of concomitant molecular alterations to HER2 amplification on the clinical outcome of trastuzumab-treated patients. Using immunohistochemistry (IHC), copy number variations (CNVs), and Ion Ampliseq Cancer Panel, we analyzed the status of concomitant alterations in 50 HER2-positive advanced GC patients treated with trastuzumab in combination with other chemotherapeutic agents. The percentage of tumor samples with at least one concomitant alteration was 40% as assessed by IHC, 16% by CNVs, and 64% by Ampliseq sequencing. Median progression-free survival (PFS) was 8.0 months (95% confidence interval, 4.8-11.3). Patients were divided into two subgroups according to PFS values with a cutoff point of 8 months; results show that concomitant genomic alterations do not correlate with trastuzumab response. However, CNVs of CCNE1 significantly correlated (p < 0.05) with a shorter survival time. Our findings indicate that additional alterations implemented for prediction of clinical benefit from HER2-targeting agents in GC remained unclear. Further studies will be needed to elucidate the role of each specific biomarker and to optimize therapeutic approaches.