Epidermal growth factor (EGF) and its receptor play critical roles in non-small cell lung cancer (NSCLC) carcinogenesis. A functional polymorphism in the EGF gene has been linked to increased cancer susceptibility. This study aimed to evaluate the role of the EGF +61A/G polymorphism as risk factors in NSCLC patients. For the present case-control study, we analyzed 112 NSCLC and 126 cancer-free controls from Portugal. Following DNA isolation from peripheral blood, EGF +61A/G polymorphism was assessed by polymerase chain reaction-restriction fragment length polymorphism. Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95 % confidence intervals (95 % CI). False-positive report probability was also assessed. The EGF +61 genotypes frequencies in NSCLC were AA (23.2 %), AG (51.8 %), and GG (25 %) and in controls, AA (40.5 %), AG (41.3 %), and GG (18.3 %). When compared to the reference genotype (EGF +61A/A), we found a statistically significant association between EGF +61 A/G (OR = 2.142, 95 % CI 1.170-3.924) and EGF +61G/G (OR = 2.398, 95 % CI 1.157-4.968) genotypes and susceptibility to development of NSCLC. Furthermore, stratification by sex revealed a trend to increased risk of males carrying +61A/G genotype for developing NSCLC (OR = 2.044, 95 % CI 0.998-4.188) when compared to A/A genotype. Our data suggest an increased risk to develop NSCLC in Portuguese population carrying the EGF +61A/G and +61G/G genotypes.