Our objective was to compare archived tissue biopsy samples from the oral cavity and oropharynx, in terms of human papillomavirus (HPV) 16 infection. We used Taqman real-time PCR assay to detect HPV16 in 121 archived biopsy samples from the oral cavity and 100 samples from the oropharynx. Among patients with oral cavity cancer (OCC), 9% (6/65) had HPV16 infection which was significantly less than those with oropharyngeal cancer (OPC) where 79% (39/50) were HPV16 positive (P < 0.001). Our results demonstrated a significant difference between genders where males had a seven times higher chance of having HPV16 infection (P < 0.001). Viral load variation between each group was demonstrated. The median viral load in OPC was similar in OCC cases, but cancer samples were significantly higher than in non-cancer cases (oral cavity samples P = 0.015; oropharynx samples P = 0.09). From our results, we conclude that there is a significant difference in HPV16 detection between OCC and OPC, and HPV16 differs greatly between male and female cancer patients. (J Oral Sci 58, 265-269, 2016)