Abstract Background Diffuse gliomas display heterogeneous biology, natural history, response to treatments, and outcome. According to the 2021 WHO Classification, an integration of histological and molecular factors is needed for the diagnosis of diffuse gliomas. The Italian Association of Neuro-Oncology (AINO), with the participation of the Italian Society of Neurosurgery (SINch), promoted a survey to explore how the 2021 WHO molecular diagnostic criteria are integrated into clinical practice in a national framework. Material and Methods A web-based survey containing 38-item multiple-choice questions was sent to members of the AINO and SINch in February 2022 via the respective email listings of these organizations. Results We collected 152 answers. Most attendants were < 45-year-old (117, 77.0%). Participants from North, Centre and South of Italy were 85 (55.9%), 38 (25.0%), and 29 (19.1%). Academic and non-academic hospitals were 35 (46.1%) and 82 (53.9%). The presence of an institutional Brain Tumour Board was reported in 108 cases (71.7%). One hundred forty attendees (92.1%) reported that IDH mutation was assessed in all glioma patients regardless of age. The 1p19q-codeletion was assessed routinely in all IDH-mutant gliomas in 88 (57.9%) or when TP53 mutation and/or ATRX expression was found (45, 29.6%). The MGMTp methylation was assessed, regardless of grading, at diagnosis in 110 (72.4%), and at second surgery in 82 (53.9%). Eighty (52.6%) performed a quantitative analysis of MGMTp status. The CDKN2A/B homozygous deletion in IDH-mutant lower-grade astrocytomas was routinely investigated in 53 (34.9%). Assessment of EGFR amplification, pTERT status or +7/-10 chromosome alterations to stratify IDH-wildtype lower-grade astrocytomas was reported in 76 (50.0%), 43 (28.3%), and 16 (10.5%) cases. Rarer alterations were less commonly investigated (H3K27M: 34, 22.4%; H3G34: 11, 7.2%; BRAF: 18, 11.8%; NTRK: 16, 10.5%), being usually evaluated in selected cases (e.g., younger patients). Academic vs non-academic hospitals treated more patients per year (> 300 in 22/70, 31.4% vs 3/82, 3.7%, p<0.001), had more available molecular technologies (53/70, 75.5% vs 37/82, 45.1, p<0.001), had a higher availability of molecular markers, such as CDKN2A/B deletion (34/70, 48.6% vs 19/82, 23.2%, p=0.001), MGMTp at second surgery (48/69, 69.6% vs 34/72, 47.2%, p=0.008), EGFR/pTERT/+7-10 (46/70, 65.7% vs 32/77, 41.6%, p=0.003), BRAF (14/70, 20.0% vs 4/82, 4.9%, p=0.002), NTRK (14/70, 20.0% vs 2/81, 2.5%, p<0.001). Conclusion The availability of new molecular markers is increasing among Italian Neuro-Oncology Centres. However, there is still a gap with the proposed criteria of the 2021 WHO Classification and the real-life application. A critical issue remains how to select patients who might benefit from the identification of some extremely rare mutations in light of targeted therapies.