Background: HPV-16 modifies the overall survival (OS) of patients with oropharyngeal cancer (OPSCC). HPV-16 has been established as risk factor for OPSCC, but HPV-16 infection may also reside in the larynx and oral cavity. We evaluated HPV-16 status on OS of Head and Neck Squamous Cell Carcinoma (HNSCC) patients. Methods: HPV-16 infection was confirmed by amplification of E6 and E7 viral oncogenes through PCR assay and E6 IHC in 185 HNSCC samples. Associations between HPV-16 status and clinicopathological parameters were performed using Fisher's exact test and x(2). Survival analysis was completed using Kaplan-Meier estimator and multivariate Cox regression analysis. Results: OS of HPV-16 positive patients was longer compared to HPV-16 negative patients (P = 0.002). HPV-16 positive tumors of the larynx (LSCC) and pharynx (PSCC) showed improved OS compared to HPV-16 negative tumors. Also, HPV-16 positive patients exposed to radiotherapy presented a better survival. Conclusions: HPV-16 status has a positive prognostic value in HNSCC. Addition of HPV-16 status to the TNM staging can provide better assessment in prognosis and guide treatment for HNSCC patients.