ROS1 has attracted much attention as a possible oncogenic driver and ROS1-rearranged tumors show sensitivity to most ALK inhibitors. We aimed to clarify the prevalence of ROS1 gene rearrangement and investigate the clinical implications of ROS1 gene copy number gain (CNG) in non-small cell lung cancer (NSCLC) patients. We carried out fluorescent in situ hybridization with ROS1 and centromere enumeration 6 probes and immunohistochemistry for ROS1 protein expression. ROS1 rearrangement was detected in 3 of 375 samples (0.8 %); all of whom were female, never-smokers, and harbored an adenocarcinoma component. ROS1 gene CNG was found in 18 cases (4.8 %). ROS1 gene CNG was significantly associated with shorter disease-free survival (DFS, 12 vs. 58 months; p = 0.003) and shorter overall survival (OS, 40 vs. 67 months; p <0.001) than the group without CNG. Multivariate analysis confirmed that ROS1 gene CNG was significantly associated with poorer DFS (hazard ratio [HR]=2.16, 95 % confidence interval [CI] = 1.22-3.81, p = 0.008), and OS ([HR] = 2.53, 95 % [CI] = 1.31-4.89, p = 0.006). ROS1 protein overexpression was observed in 5.0 % (18 out of 357), of which 2 cases harbored ROS1 gene rearrangement. There was no statistically significant correlation between ROS1 gene CNG and protein overexpression. This study demonstrated ROS1 gene rearrangement was detected in 0.8 % of surgically resected NSCLC; and ROS1 gene CNG is an independent poor prognostic factor. This survival analyses may contribute to future studies on the utility of ROS1-targeted therapy for patients.