The aim of the study was to investigate the role of high-risk human papillomavirus (HR-HPV) infection in the etiopathogenesis of oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma in non-smoking and non-drinking patients (NSNDP). Twenty-four OSCCs and 22 OPSCCs were analyzed by immunohistochemistry for p16(INK4a) protein (p16) expression and by chromogene in situ hybridization (CISH) and polymerase chain reaction (PCR) for HR-HPV DNA presence. The series included 23 males and 23 females aged 35-93 years. p16 expression was seen in 7 out of 24 (29%) OSCCs and in 22 out of 22 (100%) OPSCCs. Using CISH, HR-HPV DNA was observed in 6 out of 24 (25%) OSCCs and in 21 out of 22 (95%) OPSCCs. HPV DNA was found in 3 out of 24 (13%) OSCCs and in 18 out of 22 (82%) OPSCCs using PCR. HPV 16 and 33 were detected in 16 and in two cases, respectively. Compared with OSCCs, OPSCCs more frequently showed basaloid morphology (p < 0.0001), lymph node involvement (p = 0.0063), diffuse p16 expression (p < 0.0001), HR-HPV DNA presence using both CISH and PCR (p < 0.0001; p < 0.0001), and better outcome. The sensitivity and specificity of p16 expression for HR-HPV DNA presence detected by CISH were 0.89 and 0.95, respectively, and 0.95 and 0.85 for PCR detected HPV DNA. The sensitivity and specificity of CISH for PCR detected presence of HPV DNA were 1.00 and 0.73, respectively. Our study is the first larger study analyzing OSCC and OPSCC in NSNDP. Our results indicate that unlike OSCC, a vast majority of OPSCCs may be associated with HR-HPV infection.