Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood-derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high-dimensional single-cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border-associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease-specific transformations in the immune microenvironment during aging and in models of Alzheimer’s disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease. Display omitted •High-dimensional cytometry reveals diverse immune cells in the steady-state CNS•CD38 and MHCII distinguish CNS border-associated macrophage (BAM) subsets•A subset of microglia responds to aging and neurodegeneration•All microglia are homogenously affected in neuroinflammatory disease It has been challenging to map leukocytes in the brain, particularly during pathology. Mrdjen et al. combine high-dimensional single-cell cytometry with fate mapping to capture the immune landscape of the brain. They identify different subsets of myeloid cells and the phenotypic changes in CNS immune cells during aging and in models of Alzheimer’s disease and multiple sclerosis.