Intratumoral heterogeneity mirrors subclonal diversity and might affect treatment response. To investigate molecular heterogeneity of primary breast cancer specimens, we determined the amplification status of growth regulatory genes (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1) in macroscopically and microscopically separate areas of individual tumors (n = 21). Using laser-assisted microdissection and quantitative PCR, we found marked intratumoral heterogeneity with different patterns for each gene. Molecular heterogeneity in amplification pattern could be demonstrated between both macroscopically (0.5 to several centimeters) and microscopically (10 to several hundred micrometers) distant tumor areas. C-erbB2 amplification proved to be the most stable amplification in individual tumors, with heterogeneity occurring in only 36% of amplified cases. By contrast, amplification of c-myc and cyclinD1 revealed varying patterns in the vast majority of amplified cases (100% and 83%). The constancy of c-erbB2 amplification underlines its presumed importance in breast cancer biology. We conclude that the molecular heterogeneity of breast cancer as evidenced in this study requires thorough and representative sampling of different tumor areas when the biologic significance of somatic mutations is considered. Patterns of heterogeneity can be used to trace the clonal evolution within different compartments of an individual tumor.