Assessment of the left ventricular (LV) function by three-dimensional echocardiography (3DE) is potentially superior to 2D echo echocardiography (2DE) for LV performance assessment. However, intra- and interobserver variation needs further investigation. We examined the intra- and interobserver variability between 2 and 3DE in a general population. In total, 150 participants from the Copenhagen City Heart Study were randomly chosen. Two observers assessed left ventricular ejection fraction (LVEF), end-diastolic (EDV) and end-systolic volumes (ESV) by 2DE and 3DE. Inter-, intraobserver and intermodality variabilities are presented as means of difference (MD), limits of agreement (LoA), coefficient of correlation (r), intraclass correlation coefficients (ICC). The lowest MD and LoA and highest r- and ICC-values was generally seen among the 3D acquisitions, with the 3D EDV interobserver as the best performing estimate (r = 0.95, ICC = 0.94). The largest MD, LoA and lowest r- and ICC-values was found in the interobserver 2D LVEF (r = 0.76, ICC = 0.63. For the intraobserver analysis, there were statistically significant differences between observations for all but 3DE EDV (p = 0.06). For interobserver analysis, there were statistically significant differences between observers for all estimates but 2DE EDV (p = 0.11), 3D ejection fraction (p = 0.9), 3DE EDV (p = 0.11) and 3D ESV (p = 0.15). Three-dimensional echocardiography is more robust and reproducible than 2DE and should be preferred for assessment of LV function.