Background: The ALK, RET, and ROS1 genes are involved in gene rearrangements in a fraction of non-small cell lung cancers. The resulting oncogenic fusion genes define molecular sub-types of NSCLC with distinct sensitivities to treatment with various kinase inhibitors. We developed real-time reverse transcriptase PCR assays to detect rearrangements of ALK, RET, and ROS1 in FFPE lung cancer tissue. Methods: mRNA from NSCLC FFPE tissue samples was reverse transcribed to cDNA. Multiplex quantitative PCR was performed to detect 9 variants of EML4-ALK fusions, 9 variants of RET fusions and 14 variants of ROS1 fusions. A total of 409 samples were analyzed: 267 were classified as adenocarcinoma, 104 as squamous cell carcinoma and 38 had undetermined histology. EGFR and KRAS mutation status is unknown. The junctions of fusion-positive samples were sequenced by Sanger sequencing. Results: Among the 409 NSCLC specimens tested the frequency was 5.4% (22/409) for EML4-ALK fusions, 1.5% (6/409) for RET fusions, and 2.2% (9/409) for ROS1 fusions. EML4-ALK fusions were more prevalent in patients that were less than 60 years old (9.1% versus 2.0%, p= 0.004). The TNM stage was not correlated with the presence of any of the fusions. The table below lists the frequencies for specific rearrangements as determined by sequencing the real-time PCR products. Conclusions: Real-time PCR assays based on cDNA from FFPE tissue can identify patients with ALK, RET and ROS1 fusion genes. The ALK, RET and ROS1 assays will allow selection of patients most likely to respond to therapies that specifically target these cancer drivers. Further clinical testing of NSCLC patients in the Chinese population will be performed to support SFDA registration of these assays in China.