Epidermal growth factor receptor (EGFR) has an important role in cell proliferation, differentiation, and transformation. Several alterations and activation of EGFR have been identified in tumors. Inhibitors that impair EGFR activity have been identified and studied for cancer therapy, so the present study was conducted to comprehensively assess the amplification, mutation, and expression of EGFR in areca-associated oral squamous cell carcinoma (OSCC), which might be beneficial for targeting therapy. Gene copy number amplifications of EGFR were identified in 33% (14/42) cases of OSCC. Six cases of OSCC had a high copy number amplification. Direct sequencing of PCR products of 20 representative cases of OSCC revealed no somatic mutation in the kinase domains of EGFR. Sixty-seven percent (28/42) of the OSCC cases had nuclear and/or cytosolic EGFR immunoreactivity. Significant increases in EGFR copy number and EGFR immunoreactivity were found in OSCC subjects compared with long-term areca chewers, or compared with match adjacent oral mucosa (P<0.0001 and P=0.029, respectively). Interestingly, OSCC with nodal involvement had significantly higher EGFR gene copy number than OSCC without nodal involvement (3.194+/-0.740 versus 1.733+/-0.246; P=0.050). Our data suggest that genomic amplification could be a genetic basis underlying activation of the EGFR pathway in areca-associated OSCC.