Assessment of human epidermal growth factor receptor-2 status is standard practice in women with breast cancer. Most laboratories use immunohistochemistry as a screening test, with equivocal results confirmed by fluorescence in-situ hybridization (FISH). Chromogenic in-situ hybridization (CISH) is a relatively new method for detection of gene amplification using a peroxidase reaction, which can be viewed using a standard light microscope. This study was undertaken to validate CISH as a method for assessing human epidermal growth factor receptor-2 gene amplification. The gene amplification status of human epidermal growth factor receptor-2 immunohistochemistry negative (0/1+, n = 69; Group 1), immunohistochemistry positive (3+, n = 50; Group 2) and equivocal tumor samples (2+, n = 135; Group 3) was evaluated by FISH and CISH, and the concordance between FISH and CISH results calculated. In Group 1, 67/69 cases did not show amplification by CISH and 69/69 showed no amplification by FISH. Two cases were discordant; therefore, fluorescence/CISH concordance was 97%. In Group 2, 46/50 cases were amplified by FISH and 47/50 cases were amplified by CISH; three cases were not amplified by either method (immunohistochemistry false-positives). Only one case showed discordant FISH and CISH results, making the fluorescence/CISH concordance 98%. In Group 3, 89/135 cases were not amplified and 37/135 were amplified by both methods. Nine cases were discordant, giving a fluorescence/CISH concordance of 93%. The discordant cases were those with very low or borderline amplification with FISH. The high level of concordance between FISH and CISH seen in this study suggests that CISH may be a viable alternative to FISH for use in the human epidermal growth factor receptor-2 testing algorithm.