<p>Penile squamous cell carcinoma (PSCC) is an aggressive malignancy with a poor prognosis and limited therapeutic options. Tertiary lymphoid structures (TLSs) can support antitumour immunity, yet TLS maturation and maturation-associated stromal determinants in PSCC remain unclear. Here, we integrated hematoxylin and eosin (H&amp;E) staining, multiplex immunohistochemical (mIHC) colocalization, spatial transcriptomics, single-cell RNA sequencing (scRNA-seq), and bulk RNA sequencing (bulk RNA-seq) to profile TLS maturity and TLS-associated stromal–immune features in PSCC. TLS maturity was quantified using an AUCell germinal centre-like signature, and the presence of mature TLSs (mTLSs) coincided with improved survival. We identified a CCL21⁺ CAF subset that was preferentially enriched in mTLSs, spatially concentrated in the mTLS-core and adjacent to B-cell-rich areas; mIHC further confirmed dense CCL21⁺ACTA2⁺ CAFs near CD20⁺ aggregates. Along an inferred iTLS-to-mTLS continuum, the abundance of CCL21⁺ CAFs increased, and the expression of chemokines increased. Spatial and transcriptomic analyses further linked B-cell chemotaxis- and activation-related signatures to the CCL21-CCR7 axis, accompanied by germinal centre-like B-cell features. Clinically, higher CCL21 expression, elevated CCL21⁺ CAF signature scores, and stronger CCL21-CCR7 signatures in B cells were associated with favourable outcomes. Together, these data suggest that CCL21⁺ CAFs or <i>CCL21</i> are potential prognostic biomarkers for risk stratification and immune microenvironment profiling and highlight the CCL21-CCR7 axis as a candidate pathway for therapeutic modulation of TLS maturity in PSCC.</p> Graphical abstract <p></p>