O⁶-Methylguanine-DNA methyltransferase methylation status has a very good predictive value for benefit from alkylating agent therapy. The stratified therapy assignment of patients according to the O⁶-Methylguanine-DNA methyltransferase methylation status requires a standardized diagnostic test. A novel method detecting the promoter methylation status of O⁶-Methylguanine-DNA methyltransferase in tissue samples by a fluorescence polarization assay was developed. A pair of primers was used to amplify a 266 bp fragment in the promoter region of the O⁶-Methylguanine-DNA methyltransferase gene. Two probes specific for either methylated or unmethylated DNA labeled with different fluorophores hybridized with their target amplicons, and the hybridization increased the fluorescence polarization values. The methylation status was determined by the increased fluorescence polarization values. Ninety-seven glioma tumor samples were analyzed in parallel with the new assay and the nested gel-based methylation-specific polymerase chain reaction assay. The results of the methylation status of the fluorescence polarization assay were in good concordance with the results obtained with the nested gel-based methylation-specific polymerase chain reaction assay. The sensitivity and stability of the fluorescence polarization assay have been measured. The coefficient of variation of the reproducibility for the fluorescence polarization assay was <10%. The minimum detection level established with the fluorescence polarization assay was 20 copies/μL. The fluorescence polarization assay allowed the discrimination of the O⁶-Methylguanine-DNA methyltransferase methylation status at individual CpG sites directly in the solution without the 2-step approach with nested primers.