The HER2 protein, encoded by the ERBB2 gene, is a molecular target for the treatment of breast and gastric cancer by monoclonal antibodies or tyrosine kinase inhibitors. While intratumoral heterogeneity of ERBB2 amplification is rare in breast cancer it is reported to be frequent in bladder and colorectal cancer. To address the potential heterogeneity of the HER2 status in adenocarcinomas, squamous cell carcinomas and large cell undifferentiated carcinomas of the lung, 590 tumors were analyzed for HER2 overexpression and ERBB2 amplification using FDA-approved reagents for immunohistochemistry and fluorescence in-situ hybridization (FISH). Moderate and strong immunostaining (2+, 3+) was seen in 10% of the tumors. ERBB2 amplification was found in 17 (3%) lung cancer patients including 10 cases (2%) with high-level amplification forming gene clusters. ERBB2 amplification was significantly related to histologic subtype and tumor grade, resulting in 12% ERBB2 amplified tumors in the subgroup of high-grade adenocarcinomas. Heterogeneity was analyzed in all highly amplified tumors. For this purpose, all available tumor tissue blocks from these patients were evaluated. Heterogeneity of ERBB2 amplification was found in 4 of 10 tumors as assessed by FISH. These included two tumors with a mixture of low-level and high-level amplification and two tumors with non-amplified tumor areas next to regions with high-level ERBB2 amplification. High-level ERBB2 amplification occurs in a small fraction of lung cancers with a strong propensity to high-grade adenocarcinomas. Heterogeneity of amplification may limit the utility of anti-HER2 therapy in some of these tumors. Further attempts to assess the utility of HER2-targeting therapy in homogeneously amplified lung cancers appear to be justified.