Objective: Chromosomal instability (CIN) is a hallmark of cancer characterized by cell-to-cell variability in the number or structure of chromosomes, frequently observed in cancer cell populations and is associated with poor prognosis, metastasis, and therapeutic resistance. Breast cancer (BC) is characterized by unstable karyotypes and recent reports have indicated that CIN may influence the response of BC to chemotherapy regimens. However, paradoxical associations between extreme CIN and improved outcome have been observed. Methods: This study aimed to 1) evaluate CIN levels and clonal heterogeneity (CH) in MCF7, ZR-751, MDA-MB468, BT474, and KPL4 BC cells treated with low doses of tamoxifen (TAM), docetaxel (DOC), doxorubicin (DOX), Herceptin (HT), and combined treatments (TAM/DOC, TAM/DOX, TAM/HT, HT/DOC, and HT/DOX) by using fluorescencein situ hybridization (FISH), and 2) examine the association with response to treatments by comparing FISH results with cell proliferation. Results: Intermediate CIN was linked to drug sensitivity according to three characteristics: estrogen receptorα (ERα) and HER2 status, pre-existing CIN level in cancer cells, and the CIN induced by the treatments. ERα+/HER2? cells with intermediate CIN were sensitive to treatment with taxanes (DOC) and anthracyclines (DOX), while ERα?/HER2?, ERα+/HER2+, and ERα-/HER2+ cells with intermediate CIN were resistant to these treatments. Conclusions: A greater understanding of CIN and CH in BC could assist in the optimization of existing therapeutic regimens and/or in supporting new strategies to improve cancer outcomes.