Intratumoral heterogeneity of human epidermal growth factor receptor 2 (HER2) gene amplification has been reported to occur with variable frequencies in breast cancers. However, there have been few studies of its clinicopathological significance. We used tissue microarrays to evaluate two aspects of intratumoral heterogeneity of HER2 gene amplification: regional heterogeneity and genetic heterogeneity. We examined 96 invasive breast cancers in which HER2 amplification had been diagnosed in whole sections, and determined the clincopathological characteristics of those tumors. HER2 regional heterogeneity, defined as the existence of amplification/negative or amplification/equivocal patterns in different tissue microarray cores of a tumor, was present in 17 (18%) of the 96 cases. HER2 genetic heterogeneity, defined as the presence of tumor cells with a HER2/chromosome enumeration probe 17 ratio higher than 2.2 in 5-50% of the tumor cells, was found in 11 cases (11%), all of which showed HER2 regional heterogeneity. The cases with intratumoral heterogeneity of HER2 gene amplification were characterized by low grade or equivocal HER2 amplification and equivocal (2+) HER2 expression in whole sections. The patients with intratumoral heterogeneity of HER2 gene amplification had significantly shorter disease-free survival times than those with homogeneous HER2 gene amplification, and this effect was also evident in subgroup analysis by hormone receptor status. In multivariate analysis, intratumoral HER2 heterogeneity retained its status as an independent prognostic factor for disease-free survival. In conclusion, intratumoral heterogeneity of HER2 gene amplification is present in a subset of HER2-amplified breast cancers, especially in cases with low-grade HER2 amplification and equivocal HER2 expression, indicating a need for HER2 testing on more representative, larger tumor samples for accurate assessment of HER2 status in such cases. The patients with this heterogeneity have decreased disease-free survival, suggesting that genetic instability, and hence aberrant HER2 amplification in subclones of such tumors, may be associated with breast cancer progression.