Secondary lymphoid organs (SLOs), such as lymph nodes and tonsils, serve as an interface between the immune system and tumor cells as an initial antigen presentation site, critical in anti-tumor immune response and disease progression. For oropharyngeal cancers (OPCs) originating from palatine tonsils, we hypothesized that characterizing the immunological process occurring at the peri-tumoral tonsillar tissue will elucidate immune mechanisms for the lymphatic spread of the disease. A total of 33 patients were enrolled and subdivided into two cohorts. For cohort 1 (6 cases), gene expression profiles at the peri-tumoral lymphoid regions and tumor regions were analyzed using the GeoMx whole transcriptome atlas. In the peri-tumoral lymphoid regions, 237 genes were upregulated in metastasis-negative cases compared with metastasis-positive ones, but only one gene in tumor regions. For cohort 2 (27 cases), microarray analysis of peri-tumoral tonsillar tissues revealed 192 upregulated genes. Gene Ontology (GO) analyses revealed the significantly enriched GO terms associated with T cell activation and detected 10 hub genes according to the degree rank (PTPRC, TLR4, CD80, CD40, STAT3, CD28, CD40LG, CD44, CCR7, and IL7R). Gene set enrichment analysis combined with principal component analysis effectively sorted patients with or without lymph node metastases. These findings suggest peri-tumoral tonsils as a potential target to investigate the immune mechanisms associated with the lymphatic spread of the disease in OPCs.