OBJECTIVE To describe vertebral left atrial size (VLAS), a quantitative method to estimate left atrial (LA) size radiographically, and to determine its diagnostic value for prediction of echocardiographic LA enlargement in dogs with myxomatous mitral valve disease (MMVD) of varying severity. DESIGN Prospective observational study. ANIMALS 103 client-owned dogs with a left-sided systolic murmur. PROCEDURES For each dog, 3-view thoracic radiographs were obtained within 24 hours of an echocardiographic examination. The VLAS was measured on right and left lateral thoracic radiographs and compared with the left atrium-to-aortic root ratio acquired from short-axis (LA:AoSx) and long-axis (LA:AoLx) echocardiographic images. Left atrial enlargement was defined as an LA:AoLx ≥ 2.6 or LA:AoSx ≥ 1.6. Dogs were allocated to 4 groups on the basis of MMVD severity. RESULTS Of the 103 dogs, 15, 40, 26, and 22 were assigned to the control (no echocardiographic abnormalities), stage B1 (hemodynamically irrelevant MMVD), B2 (hemodynamically relevant MMVD), and C-D (MMVD with congestive heart failure) groups, respectively. Median VLAS, LA:AoSx, and LA:AoLx for the stage B2 and C-D groups were significantly greater than the corresponding medians for the control and stage B1 groups. There was a moderate positive correlation between VLAS and both LA:AoSx and LA:AoLx. Receiver operating characteristic analyses revealed that a VLAS ≥ 2.3 vertebrae was a useful predictor of LA enlargement. Intraobserver and interobserver agreements for VLAS measurements were high. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated VLAS was a repeatable and useful radiographic measurement for prediction of LA enlargement in dogs with MMVD.