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Simple Summary Patients with gastric cancer may present variations in the copy number of the HER2 gene in their primary tumors. The techniques used to detect these variations and HER2 overexpression render false positive and negative results with high frequency, and robust methodologies are required to assess this amplification and confidently select patients who may benefit from HER2-specific monoclonal antibody-based therapies. We addressed this issue by molecular biology techniques using DNA samples from tumor or distal tissue of gastric cancer patients. The HER2 and a control (IFNG) gene were subjected to differential (diffPCR) and quantitative PCR (qPCR). A cut-off point above which patients can be deemed positive was set based on the HER2/IFNG ratio, achieved using DNA from 30 healthy donors. Both, diffPCR and qPCR, identified the presence of somatic HER2 amplifications in 25% of patients in DNA from tumoral tissue, but not distal, paired tissue samples. Immunohistochemistry and immunofluorescence detected HER2 overexpression in tumor, but not distal, tissue of the patients previously identified as HER2+ by diffPCR and qPCR. Thus, the molecular biology-based techniques herein reported can identify patients with HER2 gene amplification and suitable for immune-based therapies. Abstract We have applied two PCR techniques, differential PCR (diffPCR) and qPCR for the identification of HER2 gene amplifications in genomic DNA of tumor and distal gastric samples from patients with gastric cancer. The diffPCR technique consists of the simultaneous amplification of the HER2 gene and a housekeeping gene by conventional PCR and the densitometric analysis of the bands obtained. We established a cut-off point based on the mean and standard deviation analyzing the DNA of 30 gastric tissues from patients undergoing non-cancer gastrectomy. diffPCR and qPCR yielded consistent results. HER2-overexpression was detected in 25% of patients and was further confirmed by immunohistochemistry and immunofluorescence. The approaches herein described may serve as complementary and reliable methods to assess HER2 amplification.