Nanoparticles' enhanced permeation and retention (EPR) variations due to tumor heterogeneity in naturally occurring brain tumors are commonly neglected in preclinical nanomedicine studies. Recent pathological studies have shown striking similarities between brain tumors in humans and dogs, indicating that canine brain tumors may be a valuable model to evaluate nanoparticles' EPR in this context. We recruited canine clinical cases with spontaneous brain tumors to investigate nanoparticles' EPR in different brain tumor pathologies using surface-enhanced Raman spectroscopy (SERS). We used gold nanoparticles due to their surface plasmon effect that enables their sensitive and microscopic resolution detection using the SERS technique. Raman microscopy of the resected tumors showed heterogeneous EPR of nanoparticles into oligodendrogliomas and meningiomas of different grades, without any detectable traces in necrotic parts of the tumors or normal brain. Raman observations were confirmed by scanning electron microscopy (SEM) and X-ray elemental analyses, which enabled localization of individual nanoparticles embedded in tumor tissues. Our results demonstrate nanoparticles' EPR and its variations in clinically relevant, spontaneous brain tumors. Such heterogeneities should be considered alongside routine preoperative imaging and histopathological analyses in order to accelerate clinical management of brain tumors using nanomedicine approaches.