Novel targeted treatment of non-small cell lung cancer (NSCLC) requires accurate classification of NSCLC as squamous cell carcinoma (SCC) and adenocarcinoma (AC). This study details the CK5/6 and TTF-1 immunoprofile of surgical resections of 45 NSCLCs (24 ACs and 21 SCCs) in tissue microarrays. All SCCs were CK5/6 positive, TTF-1 negative. 20 of 24 adenocarcinomas had the reverse pattern. In conclusion, all SCCs in this study were CK5/6 positive and TTF-1 negative, and therefore tumours that do not display this phenotype are unlikely to be SCCs. CK5/6 and TTF-1 is therefore a practical panel for the distinction between pulmonary SCC from AC in routine histopathology practice.