Aberrant DNA methylation is implicated in GBM pathobiology contributing to tumorigenicity and clinical features. Gene expression profiling classified GBM into four clinically relevant subtypes; Proneural (PN), Mesenchymal (MES), Neural (N) and Classical (CL), of which PN, MES and CL are common to glioma stem cells (GSCs). We hypothesize that the unique patterns of aberrant DNA methylation regulate subtype-associated GBM phenotypes. To test this hypothesis, we investigated whether unique novel DNA methylation signatures are distinctly associated with each of the GBM subtypes. We carried out genomewide 450K methylation array on a panel of 23 patient-derived GSCs and identified uniquely hypermethylated or hypomethylated gene probes to generate subtype-specific methylation signatures. These candidate genes also correlate to their expression based on our expression array and RNA sequencing data. Next, we performed independent analyses of 450K methylation datasets from the TCGA in corresponding GBM subtypes. Our data showed that, in each subtype, there are unique as well as common novel epigenetic signatures between GBM and GSCs. Our analyses revealed well-established PN-associated genes such as OLIG2, PDGFRA, and NOTCH and MES-associated genes including VIM, BCL2A1, and CASP8 with distinct subtype specific methylation patterns. We then validated methylation and expression of the candidate genes using bisulphite sequencing and qRT-PCR, respectively. We further determined that global demethylation induced their expression, suggesting that these genes are dysregulated due to DNA methylation. Additionally, radiation treated PN GSCs exhibited MES-associated phenotype that showed dysregulation of several genes such as VRK2 and MEGF10 and alterations in DNA methylation patterns. This suggests the roles of DNA methylation in regulating glioma subtype, radiation resistance and proneural-mesenchymal transition in GBM. We plan to elucidate the regulatory role of these candidates in GBM tumorigenicity using in vitro and in vivo assays. Our results provide new epigenetic insights in GBM subtype-specific malignancy, prognosis and therapeutic response.