Glioblastoma is the most common malignant brain tumor in adults, for which immunotherapy shows reduced efficacy. Current knowledge on immunotherapy failure is limited and detailed information about immune infiltrates in glioblastoma is urgently needed. We enrolled 34 glioblastoma patients collecting peripheral blood (PB), total tumor resection, or tumor from the necrotic area, the intermediate, and the marginal tissue through 5-aminolevulinic-acid (5-ALA) assisted surgery. T cells were evaluated for immune checkpoints and tissue residence memory (Trm) cell markers expression, and their cytokine production profile. Biological data were correlated with the patient's overall survival. Flow cytometry analysis showed a significantly higher frequency of T lymphocytes expressing PD-1, Trm markers in glioblastoma than in PB. In particular, we observed a preferential enrichment of CD8 cells expressing PD-1 and Trm markers in the intermediate and marginal tissue. T cells cytokine production resulted in increased glioblastoma compared with PB, in particular in PD-1+ cells and in the intermediate and marginal layers. These data suggest that CD103+ T-cell frequency in the core and TNF-a+CD8+ T cells in the intermediate layer influence the patient's survival. In conclusion, T cells obtained from different GBM layers showed different phenotypes and cytokines expression, suggesting new prognostic factors and supporting surgery particle strategy.