The expression of potential stem cell markers in HNSCCs was investigated to assess their potential clinical role. 69 primary, previously untreated oral (OSCC) and oropharyngeal squamous cell carcinomas (OPSCC) were enrolled; personal, clinical and follow-up data were collected. HPV infection and expression of 5 potential stem cell markers (CD44, CD133, Oct-4, Nanog, and Sox-2) were evaluated. HPV+ OPSCC showed lower expression of Nanog. The cytoplasmic expression of Nanog was associated with significantly worse prognosis in OPSCC, but not in OSCC. Sox-2 staining was more intense among OPSCCs. Sox-2 nuclear staining was associated with worse prognosis. Nanog expression was associated with HPV- OPSCC and may have a role as a surrogate diagnostic marker. In general, the expression profile of some stem cell markers in HNSCC seems to vary according to the site of origin and HPV infection. Nanog and Sox-2 may also have prognostic value.