The present study aimed to explore the etiological role of human papillomavirus (HPV) in tonsillar squamous cell carcinoma (TSCC).HPV status, including viral load and E6 variants, and the expression of P53, p16(INK4A), and FANCD2, in tissues of TSCC (n=24) and tonsillitis (n=31) were investigated.The frequency of high-risk HPV (HPV-16) in TSCCs (42%) was higher than that of tonsillitis (16%). HPV-16 genome was partially or fully integrated in all HPV-16-positive TSCCs. However, the viral genome was partially integrated in three out of five HPV-16-positive tonsillitis cases (p=0.037). HPV-16-positive TSCCs showed a higher frequency of p16(INK4A) expression than HPV-16-negative TSCCs and tonsillitis (p=0.011). Regardless of HPV status, TSCCs had a lower expression of FANCD2 than tonsillitis (p=0.008).The present study supports the etiological role of HPV-16 in the development of TSCC, and p16(INK4A) overexpression can be applied as a surrogate marker for the detection of high-risk-HPV in TSCC.