Results from several microarray-based studies have led to the identification of up-regulated expression levels of the DSG3 gene in pulmonary squamous cell carcinomas (SQCCs). The purpose of this study was to determine the role of DSG3 expression in the diagnosis of SQCCs of the lung and to compare DSG3 with p63, CK5, and CK6, as markers of squamous cell differentiation. Expression of DSG3 mRNA was evaluated in bulk laser capture microdissection-derived microarray data and by quantitative reverse transcription PCR on both SQCCs and adenocarcinomas. Expression levels of p63, CK5, and CK6 were evaluated in microarray data from the same set. An immunohistochemical study using antibodies directed against DSG3, p63, and CK5/6 was also performed. DSG3 was over-expressed in SQCCs but had very limited expression in both adenocarcinomas and non-neoplastic lungs. The microarray data showed that DSG3 had a sensitivity and specificity of 88% and 98%, respectively, in detecting SQCC versus adenocarcinoma. In comparison, sensitivity and specificity was 92% and 82% for p63, and 85% and 96% for CK5, respectively. The correlation coefficient between the microarray and immunohistochemical data for these genes was greater than or equal to 0.9. Using immunohistochemistry, sensitivity and specificity of DSG3 for lung cancers were 98% and 99%, respectively. Therefore, DSG3 can be a useful ancillary marker to separate SQCC from other subtypes of lung cancer.