Early diagnosis of chemotherapy-induced cardiotoxicity plays an important role in preventing heart failure. The main aim of our study was to assess left ventricular (LV) dyssynchrony measured by phase analysis of gated single-photon emission computed tomography (SPECT) as an early sign of cardiotoxicity after breast cancer chemotherapy. This cross-sectional study was conducted on patients with stage ≤ 3 breast cancer and no history of cardiovascular disease or diabetes. After mastectomy, the patients underwent rest gated SPECT myocardial perfusion imaging (MPI). Sixty patients with normal gated SPECT-MPI were selected and the imaging was performed after chemotherapy with doxorubicin, cyclophosphamide and paclitaxel. LV function and contractility parameters were extracted by QGS software and the results were compared with the t test method. The abnormality of at least one of the three phase analysis indices was considered as left ventricular dyssynchrony (LVD). The average LV end-systolic volume and ejection fraction (LVEF) before and after chemotherapy were (16.2 ± 8.0ml and 21.6 ± 11.6ml) and (73.4 ± 7.9% and 67.5 ± 9.2%) respectively, which showed a significant decrease (P < 0.05). In 2 patients (3.3%), the LVEF decreased to less than 50% after chemotherapy. The average parameters of left ventricular contractility before and after chemotherapy were, respectively, as follows: PHB (24.1 ± 7.5 and 33.8 ± 16.4), PSD (9.4 ± 6.1 and 5.7 ± 1.9) and entropy (28.9 ± 7.1 and 35.6 ± 9.7), which showed a significant increase (P < 0.05). LVD was observed in 14 patients (23.4%) after chemotherapy and prevalence of LVD was significantly higher in patients who had received a cumulative dose of doxorubicin of more than 400mg/m2 (P = 0.005). There was no relationship between age and body mass index with the incidence of LVD after chemotherapy (P > 0.05). Using phase analysis of gated SPECT-MPI, chemotherapy-induced LVD was seen in a significant number of patients with breast cancer, especially with a high cumulative dose of doxorubicin. LVD might indicate chemotherapy-induced cardiotoxicity before LVEF becomes abnormal.