Background Early recognition of acute sub-clinical allograft rejection is crucial after heart transplantation (HT). Right ventricular endomyocardial biopsy is the gold standard for diagnosing rejection but carries risk owing to its invasiveness. Two-dimensional ultrasound speckle tracking imaging (STI) may reflect early change of global and segmental myocardial functions. We hypothesized that STI can detect sub-clinical rejection of transplanted heart. Methods and Results A prospective study was carried out involving 17 consecutive HT patients (M:F=35:7, mean age 48.6짹16.6 year) who underwent a total 42 routine follow up endomyocardial biopsies. Conventional echocardiographic images and additional apical views in high frame rate for STI were obtained within 2 days of each biopsy. We got longitudinal peak systolic strain of each segments by automated tracking system. According to the International Society of Heart and Lung Transplantation criteria, 26 biopsies (Group A) had no rejection and 16 biopsies (Group B) had various degree of rejection which included 9, 1, 3 and 3 cases of grade 1A, 1B, 2 and 3A rejection, respectively. All echocardiographic examination showed normal ejection fraction. There were no regional wall motion abnormalities. Average longitudinal peak systolic strain (PSS) of all segments was decreased in Group B (-17.3짹2.2 vs -14.8짹1.6, p<0.001). Average longitudinal PSS of basal segments were not different in two groups (-14.2짹3.2 vs -13.1짹2.0, p=0.272), however that of mid and apical segments were significantly lower in Group B (-16.2짹2.3 vs -14.2짹1.8, p=0.004 and -20.6짹3.3 vs -16.8짹2.7, p<0.001). According to receiver operating characteristic analysis, average longitudinal PSS of all segments for <-15.75 could predict rejection with sensitivity of 78% and specificity 78%. Conclusions The present study demonstrates that longitudinal PSS, especially longitudinal PSS of mid and apical segments, as assessed by STI could have clinical value in early detecting for sub-clinical acute allograft rejection after HT noninvasively.