Anthracycline regimens are commonly used in the adjuvant setting for the treatment of breast cancer since it is considered that anthracycline-containing chemotherapy is beneficial for breast cancer patients. Nonetheless, anthracyclines carry serious side effects, including cardiotoxicity and bone marrow damage. Thus, predictive factors that identify benefit and sensitivity to these drugs are warranted in clinical practice. Topoisomerase II alpha (TOP2A) is proposed as a molecular target for anthracyclines and often amplified concurrently with human epidermal growth factor receptor 2 (HER-2) gene amplification. This study aimed to evaluate the association between TOP2A gene alterations (i.e. amplification or deletion) and oestrogen receptor (ER), progesterone receptor (PR), HER-2 and clinicopathological characteristics of breast cancer. Besides, we also investigated the relationship between TOP2A gene alterations and chromosome 17 polysomy. Two hundred twenty-seven formalin-fixed paraffin-embedded (FFPE) tissue samples collected from 2009 to 2015 were analysed. ER, PR and HER-2 protein expressions were evaluated using immunohistochemical staining. A total of 133 cases was further evaluated for their TOP2A gene alterations using dual hapten dual in-situ hybridisation (DDISH). Among these, 61.7% were HER-2 positive. TOP2A gene was co-amplified and deleted in 28% and 37.8% of the HER-2 positive tumours, respectively. TOP2A gene amplification was observed only in HER-2 positive tumours but not in HER-2 negative tumours. There were significant associations between TOP2A gene alterations and HER-2 gene, and TOP2A gene alterations and ER/PR expressions. TOP2A and HER-2 gene amplifications were frequently detected in patients with intermediate tumour size, lymph node metastases, high histological grade and invasive carcinoma, no special type (NST) phenotype. Tumours displaying chromosome 17 polysomy were frequently detected in TOP2A gene deletion compared to TOP2A amplified cases. There was significant association between TOP2A gene alterations and chromosome 17 polysomy. In conclusion, TOP2A gene alterations were associated with ER, PR, HER-2 and chromosome 17 polysomy.