Diffuse gliomas is a kind of common malignant primary brain tumor. Pseudogenes have multilayered biological function in the progression of human cancers. In this study, Differentially Expressed Pseudogenes (DEPs) between glioblastomas and non-tumor controls were found by bioinformatics analysis, of which the annexin A2 pseudogenes (ANXA2P1, ANXA2P2 and ANXA2P3) were significantly up-regulated, along with the parent gene annexin A2 (ANXA2 ). Among four glioblastoma subtypes, ANXA2P1 and ANXA2P2 were preferentially expressed in mesenchymal subtype and less expressed in proneural subtype. Meanwhile, Pearson’s correlation analysis revealed that the expression level of ANXA2 was positively correlated with ANXA2 pseudogenes expression. Then, the expression patterns of ANXA2 and its pseudogenes were validated in diffuse glioma specimens (n=99) and non-tumor tissues (n=12) by quantitative real-time PCR (qRT-PCR). Additionally, Kaplan–Meier analysis revealed that highly expressed ANXA2 and annexin A2 pseudogenes were associated with the poor survival outcome of glioma patients. Cox regression analyses suggested that ANXA2, ANXA2P1 and ANXA2P2 were the independent prognosis factors for gliomas. Furthermore, down-regulation of ANXA2 and ANXA2 pseudogenes might contribute to the improvement of patients’ survival who received chemotherapy and radiotherapy. These results demonstrated that ANXA2 pseudogenes and ANXA2 could be used as the novel biomarkers for diagnosis, prognosis and target therapy of gliomas.