Although breed-specific vertebral heart size (VHS) reference ranges have been reported, the relationship between VHS and severity of cardiac enlargement has not been clarified. The objective was to assess the influence of cardiac enlargement on VHS in Chihuahuas with myxomatous mitral valve disease (MMVD). Ten clinically normal Chihuahuas (Normal) and 97 Chihuahuas with MMVD were recruited. Chihuahuas with MMVD were classified according to the values of left atrium to aorta ratio (LA/AO) and left ventricular internal dimension in diastole normalized (LVIDDN). These dogs were allocated into 3 groups: LA1 (LA/AO < 1.6), LA2 (1.6 <= LA/AO < 2.0), LA3 (LA/AO >= 2.0), and into 2 groups: LV1 (LVIDDN < 1.7), LV2 (LVIDDN >= 1.7). Vertebral heart sizes, measured as mean +/- SD, were compared among groups. Optimal cutoff values of VHS were determined for mild (LA/AO >= 1.6, LVIDDN >= 1.7) and severe (LA/AO >= 2.0, LVIDDN >= 1.7) cardiac enlargement. Vertebral heart sizes (mean 6 SD) were Normal: 9.66 +/- 0.36, LA1: 10.13 +/- 0.64, LA2: 10.87 +/- 0.71, LA3: 11.71 +/- 0.78, LV1: 10.04 +/- 0.71, LV2: 11.21 +/- 0.78. LA2-3 had significantly greater VHS than Normal and LA1, whereas LA3 had the greatest VHS. LV2 had significantly greater VHS than Normal and LV1 and a VHS of 10.5 and 11.1 had optimal diagnostic accuracy for identifying mild and severe cardiac enlargement, respectively. In conclusion, VHS increased according to cardiac enlargement in Chihuahuas with MMVD; a VHS of 10.5 and 11.1 might be useful in evaluating the extent of cardiac enlargement.