Introduction: Epidermal growth factor receptor (EGFR) signaling pathway plays a crucial role in the development and progression of lung cancer. We searched for mutations of EGFR pathway genes in non-small cell lung cancers (NSCLCs) and analyzed their relationship with clinicopathologic features. Methods: Mutations of EGFR, ERBB2, ERBB3, ERBB4, KRAS, NRAS, BRAF, PTEN, PIK3CA, LKB1, and AKT1 genes were determined by direct sequencing in 173 surgically resected NSCLCs-56 squamous cell carcinomas (SCCs) and 117 adenocarcinomas (ACs). Results: Of the 173 NSCLCs, a total of 65 mutations were detected in 63 (36.4%) tumors-10 (17.9%) in SCCs and 53 (45.3%) in ACs. Mutations in EGFR pathway genes were significantly more frequent in women and ACs than in women and SCCs (p = 0.02 and p < 0.001, respectively). The mutations occurred in a mutually exclusive pattern. When the genes were divided into three subgroups according to their roles in the signaling cascade, mutations in the EGFR/ERBB2 and KRAS/BRAF genes were more frequent in ACs than in SCCs (p < 0.001 and p = 0.01, respectively). In marked contrast, mutations in the PIK3CA/PTEN were more frequent in SCCs than in ACs (p = 0.002). Furthermore, mutations in the PIK3CA/PTEN genes were more frequent in smokers (p = 0.04). Discussion: Our study demonstrates that mutations in each part of the EGFR pathway were associated with different clinicopathologic features in patients with NSCLCs. © 2010 by the International Association for the Study of Lung Cancer.