Abstract: Infections with mucosal High-Risk (HR) Human Papillomavirus (HPV) types are associated with a subset of Squamous Cell Carcinoma (SCC) of Upper Aerodigestive Tract (UAT). However, the prevalence of HR HPV-positive UAT cancers appears to vary in different geographical areas. To evaluate the role of HR HPV infection in Colombian population, we have characterized the prevalence of HPV16 infection and viral transcriptional activity in UAT cancers from 72 Colombian patients including tongue (n = 22), palate (n = 2) and gum (n = 2); larynx (n = 40) and esophagus (n = 6). HPV16 presence was determined by different molecular assays that allowed the evaluation of HPV16 DNA positivity, E6^E7 expression as well as the physical status of the viral genome i.e., integrated versus episomal. HPV16 DNA was detected in 35 and 30.8% of laryngeal and oral cavity cancers, respectively. In contrast, none of the esophageal cancers analyzed were HPV16-positive. Forty one percent of the UAT cancers HPV16 positives harboured the integrated form of HPV16 genome while 18.1% of the cases appeared to have both episomal and integrated viral DNA forms. E6 and E7 expression was detected in 41% of the UAT cancers independently of the HPV16 status. HPV16 DNA integration and E6 and E7 expression did not show any correlation with gender, age and type of cancer. These data provides evidence for the involvement of HPV16 in development of UAT cancers in the Colombian population.