Heart rate variability was measured in 81 Cavalier King Charles spaniels to investigate if it could be used to evaluate the severity of mitral regurgitation and to predict decompensation. Heart rate variability was assessed by the natural logarithm of the variance of the R-R intervals for 20 consecutive beats obtained from electrocardiographic recordings. Twenty-two of the dogs were clinically normal and 59 had mitral regurgitation caused by chronic valvular disease. The severity of mitral regurgitation was evaluated by echocardiography and thoracic radiography. Heart rate variability was found to be reduced (P < 0.001) among dogs with severe left atrial and ventricular dilatation and clinical signs of congestion. No significant differences in heart rate variability were found among normal dogs, dogs with only cardiac murmur, and dogs with echocardiographic evidence of slight to moderate left atrial and ventricular dilatation. Overall, an association was found between heart rate variability and left atrial to aortic root ratio and left ventricular end diastolic diameters (r = 0.72 and 0.64, respectively, P < 0.001), as well as heart and respiratory rate (r = 0.80 and 0.69, respectively, P < 0.001). Multiregression analysis showed that, in order of importance, heart rate, left atrial diameter and respiratory rate had significant effects on heart rate variability. Among these parameters, heart rate variability and left atrial diameter were found to be most efficient in separating decompensated dogs from compensated. It is concluded that heart rate variability may provide the clinician with valuable information when assessing the severity of mitral regurgitation caused by chronic valvular disease.