Gliomas consist of multiple cell types, including an abundant number of microglia and macrophages, whereby their impact on tumor progression is controversially discussed. To understand their unique functions and consequently manipulate either microglia or macrophages in therapeutic approaches, it is essential to discriminate between both cell populations. Because of the lack of specific markers, generally total body irradiated chimeras with labeled bone marrow cells were used to identify infiltrated cells within the brain. However, total body irradiation (TBI) affects the blood–brain barrier integrity, which in turn potentially facilitates immune cell infiltration. In this study, changes on the blood–brain barrier were avoided using head‐protected irradiation (HPI). Head protection and total body irradiated chimeras exhibited similar reconstitution levels of the myeloid cell lineage in the blood, enabling the comparable analyses of brain infiltrates. We demonstrate that the HPI model impeded a massive unspecific influx of donor‐derived myeloid cells into naive as well as tumor‐bearing brains. Moreover, experimental artifacts such as an enlarged distribution of infiltrated cells and fourfold increased tumor volumes are prevented in head‐protected chimeras. In addition, our data evidenced for the first time that microglia are able to up‐regulate CD45 and represent an inherent part of the CD45^high population in the tumor context. All in all, HPI allowed for the unequivocal distinction between microglia and macrophages without alterations of tumor biology and consequently permits a detailed and realistic description of the myeloid cell composition in gliomas.