6089 Background: Elevated levels of tumor-infiltrating lymphocytes (TILs) have been correlated with improved survival rates in cancer patients. Among TIL subgroups, tissue-resident memory T cells (TRM, CD8+CD103+) are recognized as pivotal contributors to the anti-cancer immune response. In this study, we aimed to evaluate TRM presence in Head and Neck Squamous Cell Carcinoma (HNSCC) tissue utilizing a developed tissue microarray (TMA) and a multiplex immunohistochemistry (MxIHC) approach. Methods: HNSCC cases from Southampton Hospital spanning the years 2000 to 2016 were reviewed, resulting in the identification of approximately 300 cases with sufficient primary tumor material. A TMA was constructed by triplicate coring of marked tumor areas on all slides. Subsequently, MxIHC, incorporating markers such as CD8 and CD103, was performed using a stain-scan-strip methodology. Digital image analysis software was employed to analyze scanned slides, and a quality check (QC) was conducted. Results: Following QC, 193 primary tumors remained (hypopharynx and larynx: n = 28, lip and oral cavity: n = 51, oropharynx: n = 114). Significantly higher counts of CD8 T cells and TRM were observed in the combined group of oropharynx, lip and oral cavity compared to the combined group of larynx and hypopharynx. This significance persisted even after excluding HPV negative oropharynx cases. HNSCC of the lip and oral cavity itself had statistically more TRM than larynx and hypopharynx. No differences were found in the subgroups between tonsil and tongue base. Stratifying the analysis by age revealed a statistically significant increase in TRM infiltration in HNSCC among young patients (≤50 years) compared to the 51-60 years and 61-70 years age groups, a pattern not observed for CD8 alone. Conclusions: This study underscores the importance of HNSCC heterogeneity and highlights the impact of age on immune infiltration, emphasizing the unique contribution of TRM in the anti-cancer immune response.