Background: The role of estrogen receptor status in the carcinogenesis of lung cancer remains elusive. A census of clonal and sub-clonal mutations has been defined through the analyses of evolutionary histories of cancers. We previously reported a prospective multicenter molecular epidemiology study (JME study; Kawaguchi T, J Clin Oncol 2016), which included the expression levels of estrogen receptors β (ER) using immunohistochemistry(IHC) and the mutational profile using next generation sequencing as well as solid smoking information and reproductive/ hormonal risk factors from the detailed questionnaire. Methods: Utilizing the data of the JME study, the impact of ER in lung cancer development was explored. All the patients underwent surgery. In 441 ever- and 435 never-smokers, ER were observed in 46.4% and 53.5%, respectively. The cancer-associated 72 gene mutations and 5 gene amplifications examined in this study included EGFR, SMAD4, APC, FBXW7, BRAF, STK11, PIK3CA, TP53, PTEN, KRAS, CTNNB1, NFE2L2, NF1, and MET. ALK fusion was detected by IHC. Patients were enrolled between July 2012 and December 2013, with follow up until November 30th, 2017.Cox proportional hazards models were used to assess the ER expression on relapse free survival (RFS) and overall survival (OS). A logistic regression model was applied to assess the impact of ER (positive vs. negative) on gene alterations, using sex, smoking history, age and stage as independent variables. Results: ER expression was significantly higher in never smokers (vs. ever smokers; p = 0.022) and earlier stage (stage I vs. II-IV; p = 0.002). Patients with ER positive tumors had a longer RFS than those with negative tumors (RFS rate at 4 years: 33.7 vs. 26.5%; p = 0.021), however, there was no significant difference in OS between the two groups (p = 0.108). In the impact of ER status on the gene alterations, mutations in EGFR (p = 0.003), TP53 (p = 0.007) and CTNNB1 (p = 0.027) were significantly associated with ER expression. Multivariate analysis showed that EGFR mutations (OR = 1.394, 95%CI: 1.029-1.890, p = 0.032) and CTNNB1 mutations (OR = 0.272, 95%CI: 0.087-0.853, p = 0.026) were significantly associated with ER expression, while there was a trend for significance with TP53 mutations (OR = 0.737, 95%CI: 0.537-1.011, p = 0.059). Conclusions: ER positive status triggered the clonal EGFR mutations and suppressed the sub-clonal mutations of TP53 and CTNNB1. It is suggested that ER plays a critical role in the alterations of EGFR/TP53/CTNNB1 axis in lung cancer evolution.