Extensive research indicates that the kinesin superfamily (KIFs) regulates tumor progression. Nonetheless, the potential prognostic and therapeutic role of KIFs in glioma has been limited. Four independent cohorts from The Cancer Genome Atlas (TCGA) database and the Chinese Glioma Genome Atlas (CGGA) database were generated into a large combination cohort for identification of the prognostic signature. Following that, systematic analyses of multi-omics data were performed to determine the differences between the two groups. In addition, IDH1 was selected for the differential expression analysis. The signature consists of five KIFs (KIF4A, KIF26A, KIF1A, KIF13A, and KIF13B) that were successfully identified. Receiver operating characteristic (ROC) curves indicated the signature had a suitable performance in prognosis prediction with the promising predictive area under the ROC curve (AUC) values. We then explored the genomic features differences, including immune features and tumor mutation status between high- and low-risk groups, from which we found that patients in the high-risk group had a higher level of immune checkpoint modules, and IDH1 was identified mutated more frequently in the low-risk group. Results of gene set enrichment analysis (GSEA) analysis showed that the E2F target, mitotic spindle, EMT, G2M checkpoint, and TNFa signaling were significantly activated in high-risk patients, partially explaining the differential prognosis between the two groups. Moreover, we also verified the five signature genes in the Human Protein Atlas (HPA) database. According to this study, we were able to classify glioma patients based on KIFs in a novel way. More importantly, the discovered KIFs-based signature and related characteristics may serve as a candidate for stratification indicators in the future for gliomas.