AbstractGliomas are the most frequent primary tumors of the brain. Glioma progression is regulated by the tumor microenvironment, which is mainly composed of tumor‐associated microglia (TA‐MG) and monocyte‐derived macrophages (MDM). Recent studies have highlighted the distinct properties of these cells in glioma progression. However, their spatiotemporal alteration during tumor progression has not been fully explored. Using a genetic lineage tracing approach, we show that TA‐MG and MDMs differ in their spatiotemporal distribution and interaction with other components of the glioma microenvironment. MDM were present only inside the tumor, whereas TA‐MG accumulated both outside and inside the tumor. However, TA‐MG was eliminated from the tumor mass as the tumor progressed. Depletion of MDM led to enhanced occupancy of TA‐MG in the tumor core, indicating that TA‐MG elimination was regulated by MDM. TA‐MG and MDM are heterogeneous cell populations whose compositions and properties can change during tumor progression. Finally, MG, TA‐MG and MDM were enriched in the perivascular area (PVA) compared to more distal blood vessel‐associated areas. However, inside the tumor, the MDM enrichment in PVA was higher than that in TA‐MG. Collectively, we established that TA‐MG and MDM exhibit different spatiotemporal features in glioma, suggesting distinctive roles during tumor progression.