Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.