The progression of ductal carcinoma in situ (DCIS) to infiltrating and metastatic cancer of the breast is thought to be a consequence of clonal expansions of neoplastic cells with progressively more genetic alterations. To study this progression, we first dissected multiple foci from each of 23 breast tumors with DCIS only and 20 cases with synchronous DCIS and infiltrating cancer. We than tested microsatellite markers by PCR for allelic losses in the individual foci for loci on chromosomes 6q, 9p, 11q, 13q, 16q, 17q, and 17p. The patterns of allelic losses identified in the in situ cancers were generally conserved in the synchronous infiltrating tumors, supporting the paradigm that the infiltrating tumors are clonally derived from the in situ lesions. However, in 8 (40%) of the 20 cases with synchronous in situ and invasive cancer, heterogeneous patterns of allelic loss at one or more chromosomal loci were observed in adjacent DCIS foci. Moreover, some of the allelic losses recognized in in situ portions of the tumors were not conserved in the clonal progression to the synchronous invasive tumor. Such allelic loss heterogeneity was noted in only 1 of the 20 infiltrating tumors and only 3 of the 23 cases of DCIS without invasion that were studied in a similar manner. This heterogeneity indicated genetic divergence during the clonal evolution of breast cancer, particularly at the time when in situ cancers progress to invasive cancers.