Abstract Diffuse gliomas, which are the most common primary brain tumors, exhibit significant variations in their characteristics such as morphology, location, genetic alterations, and response to treatment. In 2021, the World Health Organization (WHO) introduced updated guidelines for glioma classification based on molecular alterations. This study aimed to classify 476 primary diffuse glioma samples utilizing a next-generation sequencing OncoPanel AMC, targeted DNA sequencing, in accordance with the 2021 WHO criteria. The samples were divided into 3 subtypes: oligodendroglioma (ODG; n=70), diffuse astrocytoma (DA; n=65), and glioblastoma (GBM; n=341). The median age of patients diagnosed with GBM (62 years) was significantly higher compared to those with ODG (50 years) and DA (42 years) (Page=0.056 [ODG vs.DA], 3.06×10-11 [ODG vs.GBM], and 3.06×10-11 [DA vs.GBM]). Gender and tumor mutational burden did not exhibit significant associations with the subtypes. Consistent with the 2021 WHO criteria, ODG samples exhibited frequent mutations in IDH1 (91%), NOTCH1 (27%), FUBP1 (23%), CIC (20%), and IDH2 (9%). DA samples had a high prevalence of mutations in IDH1 (98%), TP53 (91%), ATRX (78%), and IDH2 (2%). In contrast, GBM samples showed mutations in IDH-wildtype and EGFR (18%). Cox multivariate regression analysis of 341 GBM patients (median survival=15.9 months) revealed that older age, lack of MGMT promoter methylation, and impaired performance status (Karnofsky performance scale< 80) were significantly associated with survival outcome (HRage=1.40, Page=7.64×10-3, HRMGMT=1.43, PMGMT=6.98×10-3, and HRKPS=1.79, PKPS=3.05×10-6). In summary, by utilizing the AMC OncoPanel and the 2021 WHO criteria, we successfully classified diffuse glioma into three distinct.