Background: It has been reported that CRKL amplification, associated with overexpression, has induced cell transformation as well as resistance to EGFR inhibitors. We analyzed CRKLamplification in archived genomic DNA samples from the Shizuoka Lung Cancer Mutation Study. Methods: To identify CRKL amplification, we performed copy number analysis using the real-time PCR system. The standard curve was generated by PCR of serially diluted plasmid clones of CRKL and a reference gene. Triplicate reactions were performed using 2 ng of genomic DNA extracted from surgically resected tissues and tumor biopsies from non-small cell lung cancer (NSCLC) patients. Biopsy samples were obtained from NSCLC patients with EGFR mutations. In surgically resected tissues, TruSeq amplicon cancer panel was used for the detection of somatic mutations in 48 cancer related genes followed by ultra-deep sequencing (Illumina) at an average coverage of approximately 3,400x. ALK, ROS1 and RET translocations and EGFR, MET, PIK3CA, FGFR1 and FGFR2 amplifications were also detected by multiplex RT-PCR and quantitative PCR, respectively. Results: Between July 2011 and March 2013, 268 NSCLC patients treated with surgical resection, and 40 NSCLC patients with EGFR mutations, were enrolled in this study. Patient characteristics (treated with surgical resection) were as follows: median age (range) 69 (38-92) years; female 35%; never-smoker 27%; histology adenocarcinoma/squamous cell carcinoma/others 74/23/3 %; and differentiation well/moderate/poor 21/53/23 %. We detected CRKL amplification in 7.5% of 268 NSCLC patients treated with surgical resection. Frequencies of CRKL amplification in patients with gene alterations were as follows: 7.5 % of 93 EGFR; none of 38 KRAS, 8.7% of 23 PIK3CA; 8.8% of 91 TP53, none of 4 EML4-ALK, and 50% of 4 KIF5B-RET. 18% of 40 patients with EGFR mutations showed CRKL amplification before EGFR-TKI treatment. Conclusions: CRKL amplification was identified in 7.5% of NSCLC patients. These results suggest that CRKL amplification may be mutually exclusive with mutations in KRAS, and frequently observed in those with KIF5B-RET.