The influence of tertiary lymphoid structures (TLSs) on disease progression and the response to immunotherapy in head and neck squamous cell carcinoma (HNSCC) is well established, yet the heterogeneity among these structures remains largely unexplored. We utilized digital spatial profiling technology to perform in situ transcriptomic sequencing of TLSs across varying levels of maturation and distinct tumor regions within HNSCC. We assessed the prognostic significance of TLS maturation and spatial distribution in 260 patients with HNSCC through hematoxylin and eosin staining and multiplex immunohistochemistry. Furthermore, we established a TLS scoring system to predict survival in patients with HNSCC. Our study revealed that mature TLSs in the intratumor region (Intra-TLSs) of HNSCC, enriched with memory B cells, plasma cells, and CD4