Tremendous progress has recently been made in the development of high-throughput microarray technologies for monitoring genetic alterations and expression profiles of genes in cancer cells. Such technological advances appear to be lacking, however, for another common molecular alteration, namely DNA methylation, in cancer. We have recently developed a novel technique, called differential methylation hybridization (DMH) that provides for the first time, an opportunity to conduct genome-based methylation analysis in breast cancer. The first part of this innovation was the generation of over 1,000 CpG island fragments (or tags) as hybridization templates arrayed onto nylon membranes. The second part involved preparation of radiolabelled amplicons, representing a pool of methylated DNA from the test genome. These amplicons were prepared from 28 paired normal and breast tumour tissues and used as probes in array-hybridization. Positive hybridization signals identified by the tumour amplicon, but not by the normal amplicon, indicate the presence of hypermethylated CpG island loci in breast tumours. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumours relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histologic grades of breast tumours. Poorly differentiated tumours appeared to exhibit more hypermethylated CpG islands than their well or moderately differentiated counterparts (P<0.041). Our early findings lay the groundwork for population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.