At recurrence of breast cancer, the therapeutic target is the metastases. However, it is current practice to base the choice of systemic treatment on the biomarker profile of the primary tumor. In the present study, confirmatory biopsies were obtained from suspected metastatic lesions and compared with the primary tumors with respect to ER, HER2, and TOP2A. In the prospective tissue-collection study, 81 patients had biopsy from a suspected relapse. Additional archived paired material was included, leaving a total of 119 patients with paired primary tumor, synchronous axillary nodes (available in 52 patients) and asyncronous metastases available for analysis. ER, HER2, and TOP2A expression of primary tumors, axillary nodes and metastases were re-analysed and determined centrally by immunohistochemistry, chromogenic in situ hybridization, and fluorescence in situ hybridization. Of the 81 patients with a biopsy from a suspected relapse, 65 (80%) were diagnosed with recurrent breast carcinoma, 3 (4%) were diagnosed with other malignancies, 6 (7%) had benign conditions, and in 7 (9%) patients the biopsy was non-representative. Discordance in ER, HER2, and TOP2A (aberration vs. normal) status between primary tumor and corresponding asynchronous metastasis was 12% (14/118), 9% (10/114), and 23% (17/75), respectively. There were no significant associations with biomarker discordance and prior adjuvant therapy, or location of biopsy. Expression of ER, HER2, and TOP2A displayed discordance with a sufficient frequency to emphasize the role of confirmatory biopsies from metastatic lesions in future management of recurrent breast cancer.