Background: Cancer/testis antigen 1B (NY-ESO-1) is exclusively expressed in various types of tumor but not in healthy normal tissue, except testis, and induces strong cellular and humoral immune responses. Therefore, it represents an ideal target for diagnostic and immunotherapeutic applications. The aim of the study, was to investigate the expression of NY-ESO-1 in oral squamous cell carcinoma (OSCC) to determine its impact as a diagnostic parameter or a therapeutic target for oral cancer. Patients and Methods: A total of 65 OSCC and 20 normal oral mucosal samples of otherwise healthy volunteers were included in this study. Expression of NY-ESO-1 was determined using reverse transcriptase polymerase chain reaction (RT-PCR). The results were Correlated to diagnosis and clinicopathological parameters. Results: NY-ESO-1 was expressed in 27.7% of the investigated tumor samples, but not in normal oral mucosal. The correlation between NY-ESO-1 expression and malignancy was significant (p=0.008). The prevalence of NY-ESO-1 expression was significantly associated with tumor size (p=0.033), but not with histological grading, positive lymph node status or clinical stage of disease. Conclusion: NY-ESO-1 expression is restricted to OSCC, clearly indicating malignancy. However, the expression rate of this antigen is too low for clinical application but it might be a useful additional biomarker within a multiple marker system for the diagnosis of OSCC. In addition, NY-ESO-1 might be a candidate for immunotherapy and polyvaccination in patients suffering front OSCC.