Abnormal DNA methylation is well established for cancer cells, but a methylation-based diagnostic test is yet to be developed. One of the problems is insufficient accuracy of cancer detection in heterogeneous clinical specimens when only a single gene is analyzed. A new technique was developed to produce a multigene methylation signature in each sample, and its potential for selection of informative genes was tested using DNA from formalin-fixed, paraffin-embedded breast cancer tissues. Fifty-six promoters were analyzed in each of 138 clinical specimens by a microarray-based modification of the previously developed technique. Specific methylation signatures were identified for atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. Informative promoters selected by Fisher's exact test were used for composite biomarker design using naïve Bayes algorithm. All informative promoters were unmethylated in disease compared with normal tissue. Cross-validation showed 72.4% sensitivity and 74.7% specificity for detection of ductal carcinoma in situ and invasive ductal carcinoma, and 87.5% sensitivity and 95% specificity for detection of atypical ductal hyperplasia. These results indicate that informative cancer-specific methylation signatures can be detected in heterogeneous tissue specimens, suggesting that a diagnostic assay can then be developed.