Introduction Cancer burden is increasing in Sub-Saharan Africa(SSA), where one-third of cancers are estimated to be caused by infectious agents. Head and neck squamous cell cancer (HNSCC) is the sixth common malignancy in SSA, including tumours in the oral cavity (OC), oropharynx(OP), hypopharynx and larynx. Tobacco and alcohol exposure are established risk factors but the role of Human Papilloma Virus (HPV) has gained recent recognition. The HPV related HNC is seen predominantly in the oropharynx, presents at a younger age and has a better prognosis. With a rapidly increasing incidence of these cancers in the developed world, it was important to study HPV in HNC in Uganda. The HPV can be detected using P16 immunohistochemistry (P16 IHC) as a surrogate marker thus making it suitable for screening. The study aimed at establishing the presence of HPV and the sites it commonly affects in upper aero-digestive tract squamous cell carcinoma (UADT SCC) at Uganda Cancer Institute (UCI) using P16 IHC. Methodology This was a cross sectional study in which 59 patients with histologically proven squamous cell carcinoma from the oral cavity, oropharynx, larynx and hypopharynx at UCI were recruited. These patients’ demographics and clinical data were collected. Tissue sections from retrieved histology samples were stained by Haematoxylin and Eosin to reconfirm SCC. Subsequently, P16 expression was determined using P16 immunohistochemistry. Results 71 patients were enrolled and 59 patients with confirmed SCC of the sites of interest were analysed. The majority (79.7%) of the participants were male and over 50 years. 59.3% were tobacco smokers, 66.1% used alcohol, 52.2% used both. Only 27.1% used none of the substances. Only 27.1% of the participants were HIV positive. Most of the tumors were in the larynx (37.3%) and 64.4% were overall TNM stage 4. The overall prevalence of HPV in UADT SCC at UCI was 20.3%, 95%CI 10.9-32.8. The oropharynx had the highest prevalence (30.8%) closely followed by the oral cavity (29.4%). Conclusion The contribution of HPV in UADT SCC at UCI using P16 IHC is significant at 20.3%. The oropharynx is the most affected site and is closely followed by the oral cavity.