Background ROS1 immunohistochemistry (IHC) using D4D6 antibody is a useful tool for screening patients with non–small cell lung cancer (NSCLC) slated for targeted therapy. Many studies and our data have identified cases that express the ROS1 protein strongly but are negative for ROS1 by fluorescent in situ hybridization (FISH). The present study investigated the driver mutation and clinicopathologic characteristics of 26 discordant cases (ROS1 IHC-positive but FISH-negative) to find new clues for distinguishing real ROS1 -rearranged cases. Patients and Methods Tumors from 26 discordant cases were analyzed for clinicopathologic characteristics, mutations in EGFR , KRAS , ERBB2 , BRAF , and PIK3CA ; fusions in ALK and RET ; and amplifications in MET , ERBB2 , and ROS1 . Results ROS1-rearranged NSCLCs were significantly more likely to be found in younger patients and at an advanced stage; they showed cribriform features, extracellular mucus, and psammoma bodies, whereas ROS1-discordant cases were found in older patients at a relatively early TNM stage and showed a lepidic growth pattern (all P 〈.001). Most of ROS1-rearranged NSCLCs were no concurrent mutation, whereas 73% of discordant cases harbored genetic aberrations, including EGFR and ERBB2 . Compared with general lung adenocarcinomas, ERBB-2 abnormality was disproportionately high in ROS1-discordant cases. Moreover, we optimized the scoring criteria for ROS1 IHC as “H score 〉150 and no concurrent mutations”; then the specificity was increased to 81.6%. Conclusions Compared with ROS1-rearranged cases, ROS1-discordant patients showed distinct clinical and morphologic features and often harbored another oncogenic driver alteration. The use of optimized screening criteria will increase the specificity of ROS1 antibody. This article is protected by copyright. All rights reserved.