OBJECTIVES: Cardiac volume measurements using three-dimensional echocardiography (3DE) have shown to deliver comparable results to magnetic resonance imaging (MRI). In a previous investigation, volume measurements of the canine left ventricle using 3DE was compared to conventional volume calculations by two-dimensional echocardiography (2DE) and M-Mode, revealing increasingly deviating results with conventional methods in the mentioned sequence. The aim of this study was to compare measurements of the left atrium and estimations of transmitral regurgitation using 3DE and 2DE in unsedated dogs. METHODS: Measurements of the left atrium were performed in 42 healthy dogs (beagles and dachshunds), using conventional linear, planimetric and volume calculations with 2DE, and volume measurements with 3DE. Agreement between methods was analyzed using Spearman's rank correlation, intraclass correlation coefficient, and Bland-Altman plot. Additionally, 20 dogs suffering from mitral valve insufficiency (CHIEF grades C2 and C3) were recruited for the estimation of transmitral regurgitation, which was approximated from 2 or 3 planes using 2DE (conventional approach), and from a live x-plane image using 3DE. RESULTS: Compared to 3DE semi-automatic volume measurements of the left atrium, volume calculations performed with 2DE (methods of discs, Simpson's rule) underestimated volumes with deviations being predictable for the respective dog sizes; whereas conventional linear and planimetric measurements allowed the most rapid investigation, but results deviated in some dogs unpredictably from those obtained with 3DE. Estimating the transmitral regurgitation using conventional 2DE was time-consuming and required replacements of the dogs during investigation, lowering compliance in the canine cardiac patients, whereas 3DE allowed accurate and more rapid evaluations without the necessity of replacing the dogs. CONCLUSIONS: Volume measurements of the left atrium are most accurate using 3DE. Additionally 3DE facilitates the estimation of transmitral regurgitation in dogs, increasing compliance with the investigation. However, further clinical and economic evaluations are needed in veterinary cardiology.