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To validate 3-dimensional (3D) 3-directional velocity-encoded (VE) magnetic resonance imaging (MRI) for flow assessment through all 4 heart valves simultaneously with retrospective valve-tracking during off-line analysis in healthy volunteers and in patients with valvular regurgitation.Three-dimensional 3-directional VE MRI was performed in 22 healthy volunteers and in 29 patients with ischemic cardiomyopathy who were suspected of valvular regurgitation and net flow volumes through the 4 heart valves were compared. Furthermore, the analysis was repeated for each valve in 10 healthy volunteers and in 10 regurgitant valves to assess intra- and interobserver agreement for assessment of respectively net flow volumes and regurgitation fraction.In healthy volunteers, the average net flow volume through the mitral valve, tricuspid valve, aortic valve, and pulmonary valve was 85 ± 20 mL, 85 ± 21 mL, 83 ± 19 mL, 82 ± 21 mL, respectively. Strong correlations between net flow volumes through the 4 heart valves were observed (intraclass correlation coefficients [ICC] 0.93–0.95) and the coefficient of variance (CV) was small (6%–9%). The repeated analysis by the same observer and by a second observer yielded good agreement for measurement of net flow volumes (ICC: 0.93–0.99 and CV: 3%–7%). Strong correlations between the net flow volumes through the 4 heart valves were also observed in the patients with valvular regurgitation (ICC: 0.85–0.95 and CV: 7%–18%). The average net flow volume through the mitral valve, tricuspid valve, aortic valve, and pulmonary valve was 63 ± 20 mL, 63 ± 20 mL, 63 ± 20 mL, 63 ± 20 mL, respectively. Furthermore, the intra- and interobserver agreement for assessment of regurgitation fraction was good (ICC: 0.86 and 0.85, CV: 12% and 13%).Flow assessment using 3D 3-directional VE MR with retrospective valve-tracking during off-line analysis enables accurate quantification of net flow volumes through 4 heart valves within a single acquisition in healthy volunteers and in patients with valvular regurgitation.