Epidermal growth factor receptor (EGFR) gene mutation and high gene copy number (CN) predict response to EGFR tyrosine kinase inhibitor therapy in the adenocarcinoma subtype of non-small cell lung cancer (NSCLC). The aims of this study were first to compare automated enzyme metallographic silver-enhanced in situ hybridization (SISH) with conventionally used fluorescence in situ hybridization (FISH) in the determination of EGFR CN in NSCLC tissue sections, and second to assess the association of EGFR CN with EGFR mutations and clinicopathological parameters. FISH and SISH were performed on tissue microarrays and large sections. Samples from 56 consecutively surgically resected NSCLC patients (cohort 1) and from 60 selected lung adenocarcinoma patients (cohort 2) were analyzed. EGFR CN was classified applying the Colorado criteria, and agreement between both methods was evaluated using κ values. EGFR CN was compared with EGFR protein expression and EGFR gene mutations. The results of SISH and FISH were identical in 114 of the 116 cases examined using the 2 techniques. One case was FISH+, SISH-, and 1 case was FISH- and SISH+. The agreement between the 2 methods was good in cohort 1 (κ=0.642 [0.428, 0.823]) and excellent in cohort 2 (κ=0.963 [0.870, 1.000]). EGFR positivity by FISH and SISH was associated with high EGFR protein expression (P<0.001) and EGFR mutation (P<0.001). These results validate the use of SISH for assessing EGFR CN alterations in NSCLC. The advantage of a permanent result and the possibility of bright-field microscopy make SISH an attractive alternative to FISH.