Purpose <p>Glioblastoma, IDH-wildtype is the most frequent and malignant primary brain tumor in adults. Tumor cells infiltrate the brain parenchyma, preventing complete resection and causing progression. Immune therapies have limited effect, but little is known about the frequency and type of immune cells in the outer part of glioblastoma, IDH-wildtype, where tumor cells start to infiltrate the brain (transition zone) and diffusely infiltrate the brain parenchyma (tumor periphery). We aimed to quantify the type and distribution of immune cells in glioblastomas, IDH-wildtype covering these areas.</p> Methods <p>We established a cohort of 54 glioblastomas, IDH-wildtype containing tissue from the tumor core, transition zone, and periphery. Patients were included if most tumor cells were positive in immunohistochemical staining for P53. Tissue sections were subject to multiplex immunohistochemistry and stained with P53 (tumor), FOXP3 (regulatory T cells), CD8 (cytotoxic T cells), and IBA1 (microglia/macrophages). A software-based classifier was trained to count the cells.</p> Results <p>The densities of CD8+, FOXP3+, and IBA1+ cells were significantly higher in the core than in the periphery and in the transition zone than in the periphery. However, the CD8+, FOXP3+, and IBA1+ cell/tumor cell ratio increased from the core to the transition zone, and the CD8+ and IBA1+ cell/tumor cell ratio increased again to the periphery. The core had the highest FOXP3+/CD8+ ratio, as well as the highest fraction of tumor cells with IBA1+ cells, CD8+ cells, and FOXP3+ cells in proximity.</p> Conclusion <p>This study highlights spatial differences in the immune microenvironment with potential implications for future immune-therapeutic strategies.</p> Graphical abstract <p></p>