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In prior work, the authors demonstrated that two-dimensional speckle-tracking (2DST) correlated well but systematically overestimated global longitudinal strain (LS) and circumferential strain (CS) compared with two-dimensional cardiac magnetic resonance tagging (2DTagg) and had poor agreement on a segmental basis. Because three-dimensional speckle-tracking (3DST) has recently emerged as a new, more comprehensive evaluation of myocardial deformation, this study was undertaken to evaluate whether it would compare more favorably with 2DTagg than 2DST.In a prospective two-center trial, 119 subjects (29 healthy volunteers, 63 patients with left ventricular dysfunction, and 27 patients with left ventricular hypertrophy) underwent 2DST, 3DST, and 2DTagg. Global, regional (basal, mid, and apical), and segmental (18 and 16 segments per patient) LS and CS by 2DST and 3DST were compared with 2DTagg using intraclass correlation coefficients (ICCs) and Bland-Altman analysis. Test-retest reproducibility of 3DST and 2DST was compared in 48 other patients.Both global LS and CS by 3DST agreed better with 2DTagg (ICC = 0.89 and ICC = 0.83, P < .001 for both; bias = 0.5 ± 2.3% and 0.2 ± 3%) than 2DST (ICC = 0.65 and ICC = 0.55, P < .001 for both; bias = −5.5 ± 2.5% and −7 ± 5.3%). Unlike 2DST, 3DST did not overestimate deformation at the regional and particularly the apical levels and at the segmental level had lower bias (LS, 0.8 ± 2.8% vs −5.3 ± 2.4%; CS, −0.01 ± 2.8% vs −7 ± 2.8%, respectively) but similar agreement with 2DST (LS: ICC = 0.58 ± 0.16 vs 0.56 ± 0.12; CS: ICC = 0.58 ± 0.12 vs 0.51 ± 0.1) with 2DTagg. Finally, 3DST had similar global LS, but better global CS test-retest variability than 2DST.Using 2DTagg as reference, 3DST had better agreement and less bias for global and regional LS and CS. At the segmental level, 3DST demonstrated comparable agreement but lower bias versus 2DTagg compared with 2DST. Also, test-retest variability for global CS by 3DST was better than by 2DST. This suggests that 3DST is superior to 2DST for analysis of global and regional myocardial deformation, but further refinement is needed for both 3DST and 2DST at the segmental level.3DST GLS and GCS agreed better with 2DTagg than 2DST.Unlike 2DST, 3DST did not overestimate GCS vs 2DTagg.Segmental strains by both 3DSTand 2DST agreed suboptimally with 2DTagg.3DST and 2DST test-retest reproducibility were similar for GLS. 3DST had better GCS test-retest reproducibility than 2DST.