We describe a high-throughput approach for investigating both copy number alterations and aberrant methylation of CpG islands in human cancer. The technique involves generation of a microarray panel of 5,150 short GC-rich tags (0.2–2 kilobases) derived from a CpG island genomic library. This DNA chip was used to study a patient with invasive breast cancer. We first restricted tumor and normal DNA from the patient with a four-base frequent cutter known to preserve larger GC-rich CpG island fragments. The digests were ligated to linkers for the polymerase chain reaction in low-amplification cycles, allowing for semiquantitative analysis in subsequent microarray hybridization. We co-hybridized amplicons from test and reference samples in a two-color fluorescence system, similar to that of the comparative genomic hybridization. The differential signal intensities observed in the microarray panel reflect copy number alterations of CpG islands in the tumor DNA. For methylation analysis, we further digested the ligated DNA with methylation-sensitive restriction enzymes and amplified it using the linker polymerase chain reaction. The amplified products may therefore contain different pools of DNA fragments owing to the differential methylation status of tumor relative to the normal control. We detected these methylation differences in subsequent hybridization using the same microarray panel. The results show that the overall changes in this tumor amount to about 16% of the CpG islands analyzed, with 8.2% of these loci showing changes in copy number, 6.1% showing changes in methylation status and 1.6% showing both types of changes. We have further categorized these genetic and epigenetic changes into eight distinct classes. Selected clones from each class have been confirmed by Southern hybridization. This study is the first such detailed analysis of complex molecular alterations in a single tumor, and it demonstrates the potential utility of this CpG island microarray for tumor classification.