Updated project metadata. Glioblastoma (GBM) is an aggressive form of brain cancer with well-established patterns of intra-tumoral heterogeneity implicated in treatment resistance, recurrence and progression. While the regional and single cell-level genetic variation of GBM have been recently resolved, downstream phenotype-level proteomic programs have yet to be assigned to specific niches. Here, we leverage laser capture microdissection and mass spectrometry-based proteomics to assign 4794 proteins to GBM’s hallmark histomorphologic niches across 20 patients. Importantly, this analysis defined 1360 regionally enriched proteins, including 502 which are proteogenomically concordant. We validate a subset of identified niche-specific markers using orthogonal immunohistochemical approaches and make the associated atlas publicly available as an online resource (https://www.brainproteinatlas.org/dash/apps/GPA). Spatial resolution of the molecular landscape of GBM, operational at the protein level, aims to further facilitate and refine our biological understanding and treatment approaches for this aggressive disease.