Epigenetic modifications have been shown to contribute to the pathogenesis of various cancers including bladder cancer. Hypermethylation in cancer is often found in CGIs in the 50 regions of genes. Although the list of aberrantly methylated genes in cancer is expanding, only a few genes so far were investigated for their usefulness as tumor biomarkers for early diagnosis and risk assessment of bladder cancer. Thus, a genome-wide screening of aberrant methylation of CGIs is needed to identify bladderspecific epigenetic markers. In this study, we performed a genome-wide screening for DNA methylation in different bladder cancer subtypes using differential methylation hybridization coupled with Agilent 244k human CpG island microarrays. We next validated the genome-wide data on a 384-plex custom Illumina Golden Gate Methylation Assay, which is based on bisulfite conversion. We found and validated a large number of possible novel epigenetic biomarkers for early detection and prognosis of bladder cancer. We also observed that subgroups of bladder tumors cluster separately due to their different methylation patterns, suggesting possible differences in pathogenesis. Our data clearly showed that the adjacent CpG loci within a CGI were comethylated. In contrast, CGIs neighboring a methylated island were usually not methylated. The data showed no bias toward promoter methylation, as the relative proportion of methylated islands in genes was similar to those encompassing promoter regions. We observed that 70% of the methylated genes are downregulated in bladder cancer. Moreover, gene body methylation also seems to be negatively correlated with gene expression. Interestingly, we found an overrepresentation of methylated genes in those genes that are the target of polycomb group protein complexes in embryonic stem cells. Further validation of selected CGIs on a separate group of bladder cancers on a custom Illumina Golden Gate Methylation assay allowed us to confirm 70% of CGIs differentiating bladder cancers from normal cells, besides validating CGIs discriminating subgroups of bladder tumors. In conclusion, this is the first genome-wide study on bladder cancer, which identified potential methylation target genes that provide putative biomarkers that may be correlated with disease course, as well as insight into the pathogenesis of bladder cancer subtypes. CLINICAL IMPACT OF GENOME-WIDE ASSOCIATION STUDIES FOR CANCER