Our purpose was to determine the accuracy of diagnosis of HER2 amplification by analysis of HER2 copy number. HER2 and chromosome 17 were measured by dual-color fluorescence in situ hybridization in breast cancer samples. At a HER2 copy number of 2 to less than 3, 16 (3.3%) of 488 cases had HER2 amplification; and at a copy number of 3 to less than 4, 32 (16.4%) of 195 cases were amplified. The proportion of cases with HER2 amplification increased considerably at HER2 copy numbers of 4 to less than 7: 50.0% at 4 to less than 5; 67.5% at 5 to less than 6, and 77.3% at 6 to less than 7. Virtually all cases were amplified at HER2 copy numbers of 7 or more. We recommend that all breast cancer cases with a HER2 copy number of 2 to 7 determined by single-color in situ hybridization should also be analyzed for chromosome 17 to obtain a more accurate diagnosis of HER2 amplification.