Radiotherapy (RT) to the thoracic region increases late cardiovascular morbidity and mortality. The impact of breast cancer laterality on cardiac function is largely unknown. The aim of this prospective study was to compare RT-induced changes in left-sided and right-sided breast cancer patients using speckle tracking echocardiography (STE). Sixty eligible patients with left-sided breast cancer and 20 with right-sided breast cancer without chemotherapy were evaluated prospectively before and early after RT. A comprehensive echocardiographic examination included three dimensional measurements and STE of the left ventricle (LV). The global longitudinal strain (GLS) was reduced from −18.3 ± 3.1 to −17.2 ± 3.3% (p = 0.003) after RT in patients with left-sided breast cancer. Similarly, regional analysis showed a reduction in the apical strain from −18.7 ± 5.3 to −16.7 ± 4.9% (p = 0.002) and an increase in basal values from −21.6 ± 5.0 to −23.3 ± 4.9% (p = 0.024). Patients with right-sided breast cancer showed deterioration in basal anterior strain segments from −26.3 ± 7.6 to −18.8 ± 8.9% (p < 0.001) and in pulsed tissue Doppler by 0.825 [0.365, 1.710] cm/s (p < 0.001). In multivariable analysis, the use of aromatase inhibitor (β = −2.002, p = 0.001) and decreased LV diastolic volume (β = −0.070, p = 0.025) were independently associated with the decrease in GLS. RT caused no changes in conventional LV systolic measurements. RT induced regional changes corresponded to the RT fields. Patients with left-sided breast cancer experienced apical impact and global decline, whereas patients with right-sided breast cancer showed basal changes. The regional differences in cardiac impact warrant different methods in screening and in the follow-up of patients with left-sided versus right-sided breast cancer.