Vasculature-induced tumor tissue heterogeneity impedes predicable drug distribution and presents notable challenges for optimizing nanoparticle (NP)-based drug delivery. However, mesoscopic-scale tumor heterogeneity across entire brain remains poorly characterized. To address this, we integrated micro-optical sectioning tomography (MOST) with high-precision three-dimensional (3D) reconstruction analysis to map pathological features of orthotopic glioma at submicron resolution across whole mice brain. Our findings uncovered significant heterogeneity in glioma invasiveness, vasculature, and compensatory angiogenesis while precisely delineating NP distribution throughout the tumor. Notably, early-stage glioma co-opted and migrated 680-micrometer upstream along the main cerebral artery within 4 days after glioma implantation. Blood-brain barrier permeability gradually increased during glioma progression, enabling NP penetrated via large-diameter vessels instead being restricted to capillaries. This work establishes a multiscale, high-resolution, 3D atlas of glioma heterogeneity and NP distribution, and bridges mesoscopic structural complexity to functional drug delivery barriers, advancing strategies to enhance oncotherapy precision in heterogeneous brain tumors.