Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the malignant transformation in gliomas. We hypothesized that quantitative analysis of methylated genes will provide prognostic values in malignant glioma patients. We used an immunocapturing approach followed by real-time polymerase chain reaction analysis to detect altered patterns of promoter methylation in O-6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, tissue inhibitor of metalloproteinase-3 (TIMP-3), and thrombospondin 1 (THBS1). The tumor tissue and paired serum as well as cerebrospinal fluid (CSF) from 66 patients with malignant gliomas were studied. Serum and CSF from 20 age-matched noncancer individuals were used as control. Promoter hypermethylation in MGMT, p16INK4a, TIMP-3, and THBS1 was detected at high frequencies in tumor tissue, serum, and CSF. None of the control serum or CSF showed aberrant methylation. Hypermethylation in serum and CSF DNA was all accompanied with methylation in the corresponding tumor tissues with 100% specificity. Highly elevated MGMT, p16INK4a, and THBS1 methylation levels in gliomas serum were the sole independent factors predicting inferior overall survival in this cohort. For progression-free survival, hypermethylation of MGMT and THBS1 in CSF were the independent prognostic factors. Multiple gene promoter hypermethylation analysis appears to be promising as a prognostic factor in glioma and as a mini-invasive tumor marker in serum and/or CSF DNA. Evaluation of these changes may help in selecting glioma patients for optimal adjuvant treatments and modifying chemotherapy.