Azanucleoside drugs such as 5-azacytidine (Vidaza) and 5-aza-2′-deoxycytidine (decitabine, Dacogen) function as DNA methyltransferase inhibitors in vitro and represent promising new drugs for the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia. In this study, we aimed to determine the effect of decitabine on the genomic methylation level in MDS patients. Comparison of different assays established micellar electrokinetic chromatography as a reliable method for the analysis of genomic methylation levels. When used for the determination of DNA methylation levels in bone marrow DNA from MDS patients during various time points of decitabine treatment, the results revealed a significant (up to 70%) demethylation in five of seven patients. Interestingly, genome-wide demethylation appeared after karyotype normalization, which suggests demethylation of nonclonal cells. Drug-induced demethylation dynamics were also confirmed by bisulfite sequencing of pericentromeric satellite elements. Our results are the first to show a genome-wide demethylating activity of decitabine in tumor material. In addition, our data uncovers novel targets of decitabine-mediated demethylation that are important for the refinement of treatment schedules with demethylating drugs.