This study was performed to compare a novel three-dimensional echocardiography (3DE) system to clinical two-dimensional echocardiography (2DE) and magnetic resonance imaging (MRI) for determination of left ventricular mass (LVM) in humans. LVM is an independent predictor of cardiac morbidity and mortality. Echocardiography is the most widely used clinical method for assessment of LVM, as it is non-invasive, portable and relatively inexpensive. However, when measuring LVM, 2DE is limited by assumptions about ventricular shape which do not affect 3D echo. Methods: A total of 25 unselected patients underwent 3DE, 2DE and MRI. Three-dimensional echo used a magnetic scanhead tracker allowing unrestricted selection and combination of images from multiple acoustic windows. Mass by quantitative 2DE was assessed using seven different geometric formulas. Results: LVM by MRI ranged from 91 to 316 g. There was excellent agreement between 3DE and MRI (r = 0.99, SEE = 6.9 g). Quantitative 2D methods correlated well with but underestimated MRI (r = 0.84–0.92) with SEEs over threefold greater (22.5–30.8 g). Interobserver variation was 7.6% for 3DE vs. 17.7% for 2DE. Conclusions: LVM in humans can be measured accurately, relative to MRI, by transthoracic 3D echo using magnetic tracking. Compared to 2D echo, 3D echocardiography significantly improves accuracy and reproducibility.