Abstract Regional diversity of anti-tumor immunity in glioblastoma (GBM) has been poorly investigated due to technical limitations. To overcome these hurdles, we developed SPTCR-seq (SPatial TCell Receptor sequencing) to spatially profile T cell immune response. Here, we performed 10X-Visium spatial transcriptomics and SPTCR-seq on 9 de-novo IDH wt glioblastoma. For SPTCR-seq, we captured enriched T-cell receptor (TCR) sequences by hybridization followed by Oxford nanopore (ONT) long-read sequencing. We established a computational pipeline for spatial demultiplexing, error correction and TCR reconstruction using a Seq2Vector model, followed by a long-term memory autoencoder to deconvolute and compare CDR3 sequence similarities across patients. The on-target rate for detected TCR genes reached over 85%, of which our long-TCR algorithm could completely reconstruct TCRs in over 70%. To decipher the regional diversity of T-cell clonality, we performed spatially weighted regression to estimate the overlap of T-cell clonal expansion to the recently described regional transcriptional niches. We found that myeloid cell infiltration was highly correlated with increased T cell diversity. We integrated single cell RNA-seq data and annotated T cell subtypes through the “natural barcode” of the CDR3 sequence, revealing that CD4+ T cells interact with myeloid cells via MHCII in segregated areas, also described as the Reactive Immune niche. In contrast, CD8+ T cells exhibit a dysfunctional/exhausted phenotype at the same location. In comparison to the myeloid-infiltrated regions, the hypoxia-associated niches showed increased CD8+ T cells with greater clonal expansion, suggesting an immune response anti-cancer immunity. Global spatial vector field analysis revealed a spatial co-occurrence of metabolic vulnerability and a mutational burden with CD8+ T effector cells negatively associated with myeloid cell infiltration. Integration of CDR3 sequences in different patients revealed higher similarity of motifs of CD4+ compared with CD8+ T cells in hypoxia areas. Our data provide new insights into the regional heterogeneity of cancer immunity.