Both mutation and amplification of epidermal growth factor receptor (EGFR) in lung cancers have been reported in association with clinical responses to tyrosine kinase inhibitors. We have reported evidence implicating mutation specifically in the "terminal respiratory unit" type of adenocarcinoma, which is characterized by expression of thyroid transcription factor 1, a lineage marker of peripheral airway cells. However, little is known about the role of gene amplification in the molecular progression of lung adenocarcinoma. In this study, we examined the topographical distribution of amplification in three microdissected portions each of 48 individual lung cancers with confirmed mutations. Relative copy number of the gene was analyzed using Taq Man-based gene dosage analysis and fluorescent in situ hybridization technique. Gene amplification was found in 11 lung cancers. Strikingly, nine of the cancers showed heterogeneous distribution, and amplification was associated with higher histologic grade or invasive growth. Because it was likely that the high-grade lesions were the origin for metastases, metastatic lymph nodes corresponding to five tumors with heterogeneous distribution were analyzed. Unexpectedly, amplification status of the metastatic sites was not always associated with gene amplification of the primary tumors, suggesting that selection of the metastatic clone may be defined by other factors. We also examined 17 precursor lesions and 21 in situ lung adenocarcinomas, and found that only one in situ carcinoma harbored gene amplification. Taken together, our results show that mutation occurs early in the development of lung adenocarcinoma, and that amplification may be acquired in association with tumor progression.