Background: The World Health Organization (WHO) classification of tumors of the central nervous system (CNS) was revised in 2016 to incorporate molecular biomarkers of importance for tumor diagnostics and clinical decision making. Thereafter, the cIMPACT-NOW consortium published a series of recommendations for the future classification of CNS tumors that have subsequently been incorporated into the new WHO classification 2021. Objectives: Which changes in the WHO classification 2021 directly affect the diagnosis and treatment of adult patients with diffuse gliomas? Materials and methods: The criteria of the WHO classification 2021 for diffuse gliomas were examined with regard to this question. Results: Mutations in the isocitrate dehydrogenase (IDH) genes 1 or 2 remain important for the classification of diffuse gliomas. Among IDH-mutant gliomas, loss of nuclear ATRX expression identifies IDH-mutant astrocytomas, while 1p/19q codeletion is diagnostic for IDH-mutant and 1p/19q-codeleted oligodendrogliomas. The nomenclature for IDH-mutant glioblastoma was changed to astrocytoma, IDH-mutant, CNS WHO grade 4. Homozygous deletion of the CDKN2A/B gene locus is a novel molecular biomarker for these tumors. IDH-wildtype diffuse astrocytomas carrying a telomerase reverse transcriptase (TERT) promoter mutation, epidermal growth factor (EGFR) gene amplification, and/or combined gains of chromosome 7 and losses of chromosome 10 (+7/−10) are now classified as IDH-wildtype glioblastomas, even when histology shows no microvascular proliferation and/or necrosis. In addition, several new pediatric-type diffuse gliomas have been introduced that must be distinguished from the more common adult-type diffuse gliomas. Conclusions: The 2021 WHO classification 2021 introduces new tumor types and implements fundamental conceptual changes based on new molecular findings, which increase diagnostic precision and improve clinical care through modified treatment recommendations. The new WHO classification also has a major impact on the design of future clinical trials in neuro-oncology.