Cancer testis antigens (CTAs) are of clinical interest as biomarkers and present valuable targets for immunotherapy. To comprehensively characterize the CTA landscape of non-small cell lung cancer (NSCLC), we compared RNAseq data of 199 NSCLC tissues to the normal transcriptome of 142 samples from 32 different normal organs. Of 232 CTAs currently annotated in the CTdatabase, 96 were confirmed in NSCLC. To obtain an unbiased CTA profile of NSCLC, we applied stringent criteria on our RNAseq data set and defined 90 genes as CTAs, of which 55 genes were not annotated in the CTdatabase. Cluster analysis revealed that CTA expression is histology-dependent and concurrent expression is common. Immunohistochemistry confirmed tissue specific protein expression of selected genes. Furthermore, methylation was identified as a regulatory mechanism of CTA expression based on independent data from the Cancer Genome Atlas. The proposed prognostic impact of CTAs in lung cancer, was not confirmed, neither in our RNAseq-cohort nor in an independent meta-analysis of 1117 NSCLC cases. Fresh frozen tumor tissue from 199 patients diagnosed with NSCLC and surgically treated 2006-2010 at the Uppsala University Hospital, Uppsala, Sweden and 19 paired normal lung tissues. Clinical data were retrieved from the regional lung cancer registry. Several of the new CTAs are poorly characterized Sample characteristics values represent; pTNM: decided by Hans Brunnström, pathologist in Lund Spring 2013 Stage according to pTNM: 1=1a 2=1b 3=2a 4=2b 5=3a 6=3b 7=IV Histology diagnosis spring 2013 HB: 1=squamous cell cancer 2=AC unspecified 3=Large cell/ NOS Surgery date: the date when sample arrived at Patologen UAS Age: age when surgery was performed Vital date: day of death or latest contact Dead: 0=no 1= yes Smoking history : 1=current 2=ex >1year 3=never WHO performance status: Performance status 0-4 Please note that the L608T_2122, L771T_1 data columns (in the processed data files) are associated with L608T and L771T samples, respectively.