BACKGROUND: p16 has often been found to be overexpressed in patients oral squamous cell carcinoma (OSCC), but its prognostic value between anatomic subsites is still unclear. OBJECTIVE: The aim of this study was to investigate the diagnostic and prognostic values of p16 in OSCC originating from tongue, gingiva or buccal mucosa. METHODS: A total of 147 OSCC patients with tumors arising from the tongue, gingiva or buccal mucosa were enrolled in this study. p16 expression was detected using immunohistochemistry (IHC), and the presence of HPV16 was determined by real-time PCR in p16 positive patients. The correlation of p16 expression with the clinical parameters was evaluated. RESULTS: Only one p16 positive patient with a cut off value of 25% and 75% was HPV16 positive. Although overall survival (OS), recurrence free survival (RFS) and metastasis free survival (MFS) had no significant differences between the p16 positive and negative patients, p16 negative patients (cut off value 25%) had more RFS in the buccal mucosa cancer (p= 0.03) than the p16-positive patients. CONCLUSIONS: The prevalence of HPV16 in Chinese OSCC patients was low. p16 overexpression decoupled from HPV infection was not a prognostic marker for OSCC patients except for patients with the buccal mucosa cancer.