Background: Detection of tumor genetic alterations is critically needed for lung cancer clinic as well as for the development of molecular targeted therapeutics. Here we report the results of a broad spectrum of genetic alterations identified in Japanese non-small cell lung cancer (NSCLC) patients by ultra-deep targeted sequencing. Methods: Highly multiplexed amplicon sequencing was performed using genomic DNA extracted from snap-frozen tumor specimens. 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 2800x. ALK, ROS1 and RET traslocations and EGFR, MET, PIK3CA, FGFR1 and FGFR2 amplifications were also detected by multiplex RT-PCR and quantitative PCR, respectively. Results: The demographics of 204 consecutive patients enrolled in this prospective study at Shizuoka Cancer Center between July 2011 and November 2012: median age 69 years (range: 38-92); male 66%; never smoker 25.5%; histology: adenocarcinoma 68.6%, squamous cell carcinoma (SQ) 27.0%, others 4.4%; tumor stage: I 53.9%, II 28.4%, III 12.7%, IV 4.9%. TP53 mutation was most frequently detected (44.4%) in all patients, particularly in SQ (67.9%). Mutations in genes such as MLH1 (4.9%), STK11 (6.3%), CTNNB1 (5.6%), SMAD4 (1.4%), VHL1 (1.4%), PTPN11 (0.7%) and GNAS (0.7%) were detected besides major mutations in genes such as EGFR (43.0%), KRAS (17.6%) and PIK3CA (14.1%) in adenocarcinoma. PIK3CA (21.4%), MLH1 (5.4%), APC (3.6%), STK11 (3.6%), FGFR2 (1.8%) and VHL (1.8%) mutations were identified in SQ and notably, 48.2% of SQ patients harbored simultaneous gene mutations, suggesting the genetic complexity of this histology. FGFR1 amplification was found in 8.9 % of SQ, suggesting lower frequency in Asian population than in Caucasian population. Conclusions: We managed to detect a wide range of genetic alterations and identified additional actionable mutations besides popular driver mutations. This approach may facilitate elucidation of detailed molecular characteristics of NSCLC, thereby implementing personalized cancer medicine.