Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Single-cell RNA sequencing (scRNA-seq) of CD11b+ myeloid cells in naive and GL261 glioma-bearing mice revealed distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrated an unforeseen molecular heterogeneity among myeloid cells in naive and glioma-bearing brains, validated selected marker proteins and showed distinct spatial distribution of identified subsets in experimental gliomas. We found higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Sex-specific gene expression in glioma-activated microglia may be relevant to sex differences in incidence and outcomes of glioma patients. ### Competing Interest Statement The authors have declared no competing interest.

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