To establish the clinical validity and accuracy of automatic thresholding and manual trimming (ATMT) by comparing the method with the conventional contouring method for in vivo cardiac volume measurements. CMR was performed on 40 subjects (30 patients and 10 controls) using steady-state free precession cine sequences with slices oriented in the short-axis and acquired contiguously from base to apex. Left ventricular (LV) volumes, end-diastolic volume, end-systolic volume, and stroke volume (SV) were obtained with ATMT and with the conventional contouring method. Additionally, SV was measured independently using CMR phase velocity mapping (PVM) of the aorta for validation. Three methods of calculating SV were compared by applying Bland–Altman analysis. The Bland–Altman standard deviation of variation (SD) and offset bias for LV SV for the three sets of data were: ATMT-PVM (7.65,
), ATMT-contours (7.85,
), and contour-PVM (11.01, 4.97), respectively. Equating the observed range to the error contribution of each approach, the error magnitude of ATMT:PVM:contours was in the ratio 1:2.4:2.5. Use of ATMT for measuring ventricular volumes accommodates trabeculae and papillary structures more intuitively than contemporary contouring methods. This results in lower variation when analyzing cardiac structure and function and consequently improved accuracy in assessing chamber volumes.