A 3D whole-tumor analysis of glioblastoma (GBM) reveals intratumoral heterogeneity and evolutionary trajectories. Using 3D surgical neuronavigation, researchers collected 103 spatially mapped samples from 10 patients with IDH-wildtype GBM. Integrative tissue and single-cell analysis identified genomic, epigenomic, and microenvironmental heterogeneity, revealing tumor-wide molecular features and regional specificity. The study inferred GBM evolution from neurodevelopmental lineage origins to genetic subclones and spatially restricted activation of tumor and microenvironmental programs. A 3D spatial map of GBM is publicly accessible, enabling 360° visualization and analysis of spatial patterns for user-selected genes and programs. The study identified patterns of GBM infiltration and clonal expansion, showing malignant cells are extensively infiltrated beyond contrast-enhancing regions. Oncogene amplifications, such as MDM4 and EGFR, were detected across tumor regions, with some showing intratumoral heterogeneity. Tumor suppressor deletions, like PTEN and CDKN2A, were also identified, reshaping both the GBM genome and epigenome. Structural variants, including chromothripsis, disrupted the genome and epigenome, driving GBM evolution. Fusion transcripts were identified, providing therapeutic opportunities for targeted inhibition or immunotherapy. Transcriptomic analysis revealed neurodevelopmental programs active tumor-wide, including those related to dividing intermediate progenitor cells and outer-radial glia. These programs provided insights into GBM lineage origins and heterogeneity. Chromatin analysis identified distinct chromatin landscapes, including those associated with neurodevelopmental and mesenchymal differentiation. Single-cell analysis revealed intratumoral heterogeneity in tumor programs, with EGFR and PDGFRA amplifications driving distinct glial sub-lineages. The study highlights the importance of 3D spatial analysis in understanding GBM heterogeneity and evolution, offering potential therapeutic targets and insights into tumor biology. The findings underscore the need for whole-tumor sampling to capture the complexity of GBM and its microenvironment, providing a framework for future research and clinical applications.A 3D whole-tumor analysis of glioblastoma (GBM) reveals intratumoral heterogeneity and evolutionary trajectories. Using 3D surgical neuronavigation, researchers collected 103 spatially mapped samples from 10 patients with IDH-wildtype GBM. Integrative tissue and single-cell analysis identified genomic, epigenomic, and microenvironmental heterogeneity, revealing tumor-wide molecular features and regional specificity. The study inferred GBM evolution from neurodevelopmental lineage origins to genetic subclones and spatially restricted activation of tumor and microenvironmental programs. A 3D spatial map of GBM is publicly accessible, enabling 360° visualization and analysis of spatial patterns for user-selected genes and programs. The study identified patterns of GBM infiltration and clonal expansion, showing malignant cells are extensively infiltrated beyond contrast-enhancing regions. Oncogene amplifications, such as MDM4 and EGFR, were detected across tumor regions, with some showing intratumoral heterogeneity. Tumor suppressor deletions, like PTEN and CDKN2A, were also identified, reshaping both the GBM genome and epigenome. Structural variants, including chromothripsis, disrupted the genome and epigenome, driving GBM evolution. Fusion transcripts were identified, providing therapeutic opportunities for targeted inhibition or immunotherapy. Transcriptomic analysis revealed neurodevelopmental programs active tumor-wide, including those related to dividing intermediate progenitor cells and outer-radial glia. These programs provided insights into GBM lineage origins and heterogeneity. Chromatin analysis identified distinct chromatin landscapes, including those associated with neurodevelopmental and mesenchymal differentiation. Single-cell analysis revealed intratumoral heterogeneity in tumor programs, with EGFR and PDGFRA amplifications driving distinct glial sub-lineages. The study highlights the importance of 3D spatial analysis in understanding GBM heterogeneity and evolution, offering potential therapeutic targets and insights into tumor biology. The findings underscore the need for whole-tumor sampling to capture the complexity of GBM and its microenvironment, providing a framework for future research and clinical applications.