Background Studies on pediatric heart transplantation (HTx) are uniquely challenging because pediatric HTx center volumes are generally low. And, the biventricular function plays an important role in the prognosis of pediatric HTx. The primary aim of our study was to evaluate biventricular function of pediatric HTx by three-dimensional speckle tracking echocardiography(3D-STE). Methods We enrolled 30 clinically well pediatric HTx patients and 30 sex- and age- matched healthy controls. All subjects underwent comprehensive echocardiographic examinations. Left ventricular (LV) global longitudinal strain (GLS), global circumferential strain (GCS), LV and right ventricular (RV) ejection fraction (EF) and RV longitudinal strain (RVLS) of free wall and septum were acquired by 3D-STE. And the correlations between strains and clinical data were explored. Results Compared with controls, LV GLS was decreased in pediatric HTx patients (P<0.05), while LV GCS and LVEF showed no difference. RVEF, RVLS (free wall) and RVLS (septum) in HTx group were diminished (P<0.05), but RVEF was still in normal range. Cold ischemic time was correlated inversely with LV GLS (β=-0.401, P<0.05). The mean pulmonary artery pressure (β=0.447, P<0.05) and postoperative tricuspid regurgitation pressure (β=0.607, P<0.05) were associated with RVLS (free wall). Conclusion Biventricular longitudinal systolic function rather than global systolic function was impaired after HTx. 3D STE may be able to evaluate the ventricular function better. Prolonged ischemic time leads to impaired LV longitudinal systolic function in pediatric HTx patients. It’s interesting that in HTx patients, it shows compensatory enhancement due to increased pulmonary vascular resistance.