Estrogens play a pivotal role in the development and progression of non-small lung cancer (NSCLC). With the discovery of estrogen receptor beta (ER beta) isoforms, some controversial roles of ER beta were explained adequately in NSCLC. In this study, our aim is to elucidate expression, distribution, and prognostic significance of ER beta 1, 2, 5 in NSCLC. Estrogen receptors beta 1, 2, 5 protein expression were confirmed by Western-blot analysis in all frozen tissues, and immunohistochemistry (IHC). Nuclear and cytoplasmic staining was evaluated and correlated with histopathologic characteristics, overall survival (OS) and disease-free survival (DFS) via Pearson chi (2) square, Kaplan-Meier plots and Cox proportional hazard models. ER beta 1 was commonly found in the cytoplasm and was the most abundant isofroms followed by ER beta 2 and ER beta 5 which were localized in the cytoplasm and nucleus. In contrast to BPL, both in nucleus and cytoplasm, ER beta 1, ER beta 2, and ER beta 5 were over expressed all in NSCLC (P < 0.05). IHC results were correlated with pathological and clinical follow-up data to delineate the distinct roles of ER beta 1, ER beta 2, and ER beta 5 in NSCLC. nER beta 1 "nuclear", cER beta 2 and cER beta 5 "cytoplasm" were in a negative correlation with the pathological stage and lymph node metastasis. In a Kaplan-Meier analysis, the expression cER beta 2 and cER beta 5 identified a group of patients with the longest DFS and OS. Cox proportional hazard models revealed that cER beta 2 and cER beta 5 predicted long time to DFS and OS. This is the first study to uncover the expression of ER beta 1, ER beta 2, and ER beta 5, and show that they were over expressed in NSCLC. Meantime, we find that positive expression of cER beta 2 and cER beta 5 were in a positive correlation with DFS, and have prognostic values for the progression of NSCLC.