ROS1 gene-rearrangement in non-small-cell lung cancer (NSCLC) patients has recently been identified as a driver gene and benefited from crizotinib treatment. However, no data are available for ROS1-positive NSCLC about chemotherapeutic options and prognostic data. We investigated pemetrexed-based treatment efficacy in ROS1 translocation NSCLC patients and determined the expression of thymidylate synthetase (TS) to provide a rationale for the efficacy results. We determined the ROS1 status of 1750 patients with lung adenocarcinoma. Patients' clinical and therapeutic profiles were assessed. In positive cases, thymidylate synthetase (TS) mRNA level was performed by RT-PCR. For comparison, we evaluated the TS mRNA status and pemetrexed-based treatment efficacy from 170 NSCLC patients with anaplastic lymphoma kinase (ALK) translocation (n = 46), EGFR mutation (n = 50), KRAS mutation (n = 32), and wild-type of EGFR/ALK/ROS1/KRAS (n = 42). Thirty-four ROS1 translocation patients were identified at two institutions. Among the 34 patients, 12 with advanced stage or recurrence were treated with pemetrexed-based first-line chemotherapy. The median progression-free survivals of pemetrexed-based first-line chemotherapy in ROS1 translocation, ALK translocation, EGFR mutation, KRAS mutation, and EGFR/ALK/ROS1/KRAS wild-type patients were 6.8, 6.7, 5.2, 4.2, and 4.5 months, respectively (P = 0.003). The TS mRNA level was lower in patients with ROS1-positive than ROS1-negative patients (264 ± 469 × 10 vs. 469 ± 615 × 10 , P = 0.03), but similar with ALK-positive patients (264 ± 469 × 10 vs. 317 ± 524 × 10 , P = 0.64). Patients diagnosed with ROS1 translocation lung adenocarcinoma may benefit from pemetrexed-based chemotherapy. TS mRNA level enables the selection of therapeutic options for ROS1 translocation patients.