The latest 2021 WHO classification redefines glioblastoma (GBM) as the hierarchical reporting standard by eliminating glioblastoma, IDH-mutant and only retaining the tumor entity of “glioblastoma, IDH-wild type.” Knowing that subclassification of tumors based on molecular features is supposed to facilitate the therapeutic choice and increase the response rate in cancer patients, it is necessary to carry out molecular classification of the newly defined GBM. Although differentiation trajectory inference based on single-cell sequencing (scRNA-seq) data holds great promise for identifying cell heterogeneity, it has not been used in the study of GBM molecular classification. Single-cell transcriptome sequencing data from 10 GBM samples were used to identify molecular classification based on differentiation trajectories. The expressions of identified features were validated by public bulk RNA-sequencing data. Clinical feasibility of the classification system was examined in tissue samples by immunohistochemical (IHC) staining and immunofluorescence, and their clinical significance was investigated in public cohorts and clinical samples with complete clinical follow-up information. By analyzing scRNA-seq data of 10 GBM samples, four differentiation trajectories from the glioblastoma stem cell-like (GSCL) cluster were identified, based on which malignant cells were classified into five characteristic subclusters. Each cluster exhibited different potential drug sensitivities, pathways, functions, and transcriptional modules. The classification model was further examined in TCGA and CGGA datasets. According to the different abundance of five characteristic cell clusters, the patients were classified into five groups which we named Ac-G, Class-G, Neo-G, Opc-G, and Undiff-G groups. It was found that the Undiff-G group exhibited the worst overall survival (OS) in both TCGA and CGGA cohorts. In addition, the classification model was verified by IHC staining in 137 GBM samples to further clarify the difference in OS between the five groups. Furthermore, the novel biomarkers of glioblastoma stem cells (GSCs) were also described. In summary, we identified five classifications of GBM and found that they exhibited distinct drug sensitivities and different prognoses, suggesting that the new grouping system may be able to provide important prognostic information and have certain guiding significance for the treatment of GBM, and identified the GSCL cluster in GBM tissues and described its characteristic program, which may help develop new potential therapeutic targets for GSCs in GBM.