Estimating the type-specific prevalence of human papillomavirus (HPV) in head and neck cancer (HNSCC) is helpful in predicting the impact of HPV immunization. To estimate the overall prevalence, and gender and age-specific prevalence of HPV in HNSCC. This cross sectional retrospective study was carried out in four pathology laboratories of Medellin, Colombia. HPV testing was performed by GP5+/6+ PCR-based RLB and HPV 16 and 18 type-specific PCR. 175 primary HNSCC cases consecutively diagnosed between 1999 and 2008 with confirmed diagnosis and amplifiable DNA were included. Overall HPV prevalence was 18.9%. HPV was found in 23.9%, 17.5% and 13.3% of the oral cavity, larynx and oropharynx cases respectively. Among HPV positive cases, 82% were HPV 16 and 18% were HPV 18. No other HPV genotypes were identified. Most patients were males. Male patients were younger that their female counterparts, particularly in oral cavity cancer cases. HPV 16 and 18 genotypes were found in nearly 20% of HNSCC cases in Colombian patients. The impact of HPV vaccination for the prevention of HNSCC in this population deserves further evaluation.