Background: We have previously shown that human epidermal growth factor receptor 2 (HER2, ERBB2) mutation and amplification represent distinct molecular entities in lung cancers (Li J Thorac Oncol 2015). The optimal method for assessing HER2 status in lung cancers for targeted therapy remains undefined. Methods: Prospective next generation sequencing (NGS) was performed on lung adenocarcinoma specimens by Memorial Sloan Kettering-Integrated Mutation Profiling for Actionable Cancer Targets (MSK-IMPACT) for potentially actionable drivers across 410 cancer related genes in 2014-2015 encompassing HER2 mutation (indels and point mutations) and amplification (fold change ≥ 1.5). Specimens found to have HER2 mutations or amplification were further assessed by fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC). HER2 IQFISH pharmDx, Dako probe was used for FISH with HER2/CEP17 ratio ≥ 2.0 defined as positive. The 4B5 Ventana antibody was used for HER2 protein overexpression by IHC. Results of the various HER2 testing were compared. Results: A total of 776 lung adenocarcinomas underwent NGS and found 21 cases (3%) of HER2 mutation and 20 cases (3%) of HER2 amplification; only one case showed concurrent mutation and amplification (S310F, fold change 11.7). 12 mutant cases (57%) harbored an identical 12bp insertion in exon 20 (p.A775_G776insYVMA). FISH analysis of 34 samples (18 mutant, 14 amplified) was positive in 1 of 18 mutant and 14 of 14 NGS amplified cases. IHC analysis of 18 mutant cases ranged from 0 to 2+, and analysis of 12 amplified cases showed high correlation with IHC 3+. All cases of HER2 amplification by FISH or high level HER protein overexpression (IHC 3+) were captured by NGS. Conclusions: This independent study confirmed that HER2 mutation and HER2 amplification are distinct molecular targets in lung cancers. IHC and FISH are suitable methods for identifying HER2 amplification but are not surrogates for assessing HER2 mutation. NGS identified HER2 mutation and all cases of HER2 amplification or high level HER2 overexpression, and constitutes an ideal method of upfront screening for all HER2 alterations for selecting patients for HER2 targeted therapy.