HPV plays a role in the development of a portion of head and neck squamous cell carcinoma (HNSCC), but only limited information on its role in southern Chinese population is available. A multicenter case-control study was conducted. HPV type, viral integration, E6/7 mRNA expression status, and TP53 mutation were determined. A total of 228 HNSCC were recruited including 137 (60.1%) oral SCC, 34 (14.9%) oropharyngeal SCC, 31 (13.6%) laryngeal SCC, 21 (9.2%) hypopharyngeal SCC, and 5 (2.2%) lip and paranasal sinus SCC. High-risk HPV infection was found in 7.5% (17/228) of HNSCC, but only a small proportion of samples had evidence of viral integration (5.3%, 12/228) or E6/7 mRNA expression (4.4%, 10/228). HPV infection with oncogenic phenotype (integration and E6/7 mRNA expression) was significantly more common in oropharyngeal SCC than controls (9/34, 26.5% vs. 0/42, 0.0%, P < 0.001). Smoking showed a significant association with HNSCC, oropharyngeal SCC, and laryngeal SCC. TP53 mutation was associated with HNSCC (P < 0.001). Older age, TP53 mutation, and HPV16 infection with oncogenic phenotypes were independently associated factors for HNSCC with odds ratios of 1.03 (1.02–1.05), 3.38 (1.71–6.66), and 9.19 (1.13–74.68), respectively. High-risk HPV infection of head and neck mucosa is not uncommon in the Hong Kong population. This study found that 26–30% of oropharyngeal carcinoma was associated with HPV infection, mostly HPV16, and that smoking which predisposes to TP53 mutations was another important risk factor. J. Med. Virol. 88:877–887, 2016. © 2015 Wiley Periodicals, Inc.