Diaphanous-related formin (DIAPH) 3 is a key regulator of fundamental processes such as division, adhesion, motility and polarity. Diaph3 is exclusively expressed in neural progenitors in the developing mouse brain and its deficiency causes aberrant cell division and aneuploidy, resulting in the loss of neural progenitor cells. Errors in mitosis often lead to mitotic catastrophe and subsequent cell death, impeding the proliferation of aneuploid cells. Nevertheless, aneuploidy is a hallmark of highly proliferative cancer cells and is generally associated with poor prognosis and therapeutic resistance. Glioblastoma, the most common and aggressive malignant primary brain tumor in adults, is characterized by a high degree of aneuploidy which is believed to drive therapeutic resistance. Methylation of the MGMT promoter, which predicts the response to temozolomide, is a well-established prognostic marker for glioblastoma. However, there is still an important level of variability in terms of survival within the MGMT-methylated group, emphasizing the need for additional predictive factors. Here, by studying glioblastoma samples, we uncovered a positive correlation between DIAPH3 expression and patient survival, which is more prominent in MGMT-methylated glioblastoma. We found that 36% and 1% of the glioblastoma samples exhibit copy number alterations and mutations in DIAPH3 respectively. DIAPH3 expression overlaps with KI67 expression in glioblastoma proliferating cells including neural progenitor-like, oligodendrocyte progenitor-like and astrocyte-like states. Finally, we identify three CpG sites in the DIAPH3 promoter that could partially regulate its expression. Combining the DIAPH3 expression with MGMT methylation could offer a better prediction of survival for patients with MGMT-methylated glioblastoma.