To investigate the value of tissue Doppler velocities for ruling out treatment-requiring acute cellular rejection (TR-ACR), in the context of myocardial deformation analysis performed by means of speckle tracking echocardiography. We performed serial echocardiograms in 37 heart transplant recipients in their first year post-transplantation within 3 h of the routine surveillance endomyocardial biopsies (EMB). The association of the sum of lateral mitral annulus systolic (s′) and early diastolic (e′) velocities, in absolute values, measured by tissue Doppler echocardiography (s′+ e′), with TR-ACR (ACR grade ≥ 2R) was investigated by multivariate analysis, including classic echocardiographic parameters and myocardial deformation variables. A total of 251 pairs of EMB and echo exams were performed, 35 (14%) with rejection grade ≥ 2R (TR-ACR). s′ + e′ was independently associated to TR-ACR (OR 0.80, 95%CI 0.72–0.89, p < 0.0005), with a C statistic of 0.79 (95%CI 0.71–0.87, p < 0.0005) by ROC curve analysis. An  s′+ e′ value ≥ 23 cm/s, present in 43% of studies, had a negative predictive value of 98% for ruling out TR-ACR. Moreover, in the same patients, s′+ e′ significantly decreased when TR-ACR occurred after a study without this condition (− 3.7 ± 3.3 cm/s, p = 0.003), but it was similar when rejection status was the same in the present versus the previous study. A drop in s′+ e′ value < 2.7 cm/s from the previous echocardiogram, had a 99% negative predictive value for ruling out TR-ACR. Tissue Doppler velocities, a widely available echo parameter, were found to be a valuable marker for ruling out TR-ACR in this multivariate study which included myocardial deformation variables.