BACKGROUND Around 20-40% of heart transplant patients experience moderate to severe rejection within the first year after heart transplantation. Endomyocardial biopsy (EMB) is a gold standard for diagnosing heart transplant rejection. There is a need for non-invasive alternatives that allow for early, safe and reliable diagnosis of acute graft rejection prior to the onset of clinical symptoms. AIMS Our aim was to investigate the potential of speckle tracking derived strain analysis in the diagnosis of acute graft rejection. METHODS Patients indicated for EMB consented to a trans-thoracic echocardiography examination (TTE) within 2 hours of the EMB. Of this cohort, those with at least 2 EMBs separated ≥ 1 week, and whose TTE could be analyzed for strain, were included. The relationship between strain and EMB results was evaluated. RESULTS Of the 43 patients included (mean age 51.33±1.79, 67% male), 23 had findings of rejection identified on at least one EMB and at least one EMB without rejection for comparison. A significant deterioration in the longitudinal strain during rejection compared to non-rejection was found on apical 4-chamber views (-11.51±0.91 vs -13.48±0.96, P=0.025) and apical 2-chamber views (-11.84±0.78 vs -14.43±0.83, P=0.002). In the patients in whom no rejection was identified on either EBM, there was no significant change in longitudinal strain values at two different time points. CONCLUSION Worsening of longitudinal strain was associated with acute cellular rejection. Routine TTE-based strain analysis could help in early detection of cardiac rejection and timing of EMB.