Although multiple echocardiographic methods exist to calculate cardiac output (CO), they have not been validated in mice using a reference method.Echocardiographic and flow probe measurements of CO were obtained in mice before and after albumin infusion and inferior vena cava occlusions. Echocardiography was also performed before and after endotoxin injection. Cardiac output was calculated using left ventricular volumes obtained from an M-mode or a two-dimensional view, left ventricular stroke volume calculated using the pulmonary flow, or estimated by the measurement of pulmonary velocity time integral (VTI).Close correlations were demonstrated between flow probe-measured CO and all echocardiographic measurements of CO. All echocardiographic-derived CO overestimated the flow probe-measured CO. Two-dimensional image-derived CO was associated with the smallest overestimation of CO. Interobserver variability was lowest for pulmonary VTI-derived CO.In mice, CO calculated from two-dimensional parasternal long-axis images is most accurate when compared with flow probe measurements; however, pulmonary VTI-derived CO is subject to less variability.