Abstract Purpose: High numbers of tumor infiltrating lymphocytes (TIL) are linked to better survival in cancer patients. TRM(CD8+CD103+) are recognized as a key player of anti-cancer immune response. To assess TRM in primary, metastatic and recurrent HNSCC we developed a tissue microarray (TMA) and used multiplex immunohistochemistry (MxIHC). Experimental Design: 379 HNSCC cases from Southampton Hospitals (2000-2016) with material of primary tumors were retrieved. Of these, 105 cases had lymph node metastases and 82 recurrences. A TMA was generated with triplicate cores for each sample. MxIHC with a stain and strip approach was performed using CD8, CD103, TIM3. Scanned slides were analyzed (digital image analysis) and quality checked (QC). Results: After QC, 194 primary tumors, 76 lymph node metastases and 65 recurrences were evaluable. Alcohol drinking was statistically significantly correlated with a reduction of TRM cells in primary tumors (no drinker vs. heavy drinker: p = 0.0036). The known survival benefit of TRM infiltration in primary tumors was not found for lymph node metastasis. In recurrences a high TRM number led to a favorable outcome after 12 months. The checkpoint molecule TIM3, was expressed significantly higher on TRM and non-TRM cells in the lymph node compared to primary tumors (p < 0.0001), which was also seen in recurrences (p 0.0134, p=0.0007, respectively). Conclusions: We confirm the prognostic impact of TIL in primary tumors and in recurrences. TRM cell density in lymph node metastases was not linked to outcome. The role of TIM3, as a therapeutic target remains to be defined.