Molecular targeted therapy is expected to be a promising therapeutic approach for the treatment of esophageal squamous cell carcinoma (ESCC); however, the gene amplification status of molecular targeted genes in ESCC remains largely unclear. The gene amplification of EGFR, HER2, FGFR2 and MET was examined using a real-time PCR-based copy number assay of 245 ESCC surgical specimens of formalin-fixed, paraffin-embedded samples. Fluorescence in situ hybridization (FISH) and comparative genomic hybridization analyses verified the results of the copy number assay. EGFR mutation was detected using the Scorpions-ARMS method. The EGFR status and drug sensitivity to an EGFR tyrosine kinase inhibitor was then evaluated in vitro. Gene amplification of EGFR and HER2 was observed in 7% (16/244) and 11% (27/245) of the ESCC specimens. A multivariate analysis revealed that HER2 amplification was a significant predictor of a poor prognosis in patients with stage III post-operative ESCC. The L861Q type of EGFR mutation with hypersensitivity to EGFR tyrosine kinase inhibitor was found in one of the eight ESCC cell lines and one del745 type of EGFR mutation was identified in 107 clinical samples. In addition, we demonstrated for the first time that FGFR2 amplification was observed in 4% (8/196) of the ESCC specimens. MET amplification was observed in 1% (2/196). In conclusion, the frequent gene amplification of EGFR, HER2 and FGFR2 and the presence of active EGFR mutations were observed in ESCC specimens. Our results strongly encourage the development of molecular targeted therapy for ESCC.