There is no established method to assess the PD-L1 expression in brain tumours. Therefore, we investigated the suitability of affibody molecule (Z_PD-L1) radiolabelled with F-18 (Al¹⁸F) and Ga-68 to measure the expression of PD-L1 in xenograft mouse models of GBM. Mice bearing subcutaneous and orthotopic tumours were imaged 1 h post-radioconjugate administration. Ex vivo biodistribution studies and immunohistochemistry (IHC) staining were performed. Tumoural PD-L1 expression and CD4+/CD8+ tumour-infiltrating lymphocytes were evaluated in human GBM specimens. Z_PD-L1 was radiolabelled with radiochemical yields of 32.2 ± 4.4% (F-18) and 73.3 ± 1.8% (Ga-68). The cell-associated radioactivity in vitro was consistent with PD-L1 expression levels assessed with flow cytometry. In vivo imaging demonstrated that ¹⁸F-AlF-NOTA-Z_PD-L1 can distinguish between PD-L1 high-expressing tumours (U87-MGvIII) and PD-L1-negative ones (H292_PD-L1Ko). The radioconjugate was quickly cleared from the blood and normal tissues, allowing for high-contrast images of brain tumours as early as 1 h post-injection. ⁶⁸Ga-NOTA-Z_PD-L1 showed heterogeneous and diffuse accumulation that corresponded to the extensively infiltrating GCGR-E55 tumours involving contiguous lobes of the brain. Lastly, 39% of analysed GBM patient samples showed PD-L1+ staining of tumour cells that was associated with elevated levels of CD4+ and CD8+ lymphocytes. Our results suggest that the investigated radioconjugates are very promising agents with the potential to facilitate the future design of treatment regimens for GBM patients.