Cardiac biomarkers are elevated in dogs with heart diseases including mitral valve disease (MVD). However, the difference in the regulation of atrial natriuretic peptide (ANP), N-terminal proANP (NT-proANP), N-terminal proB-type natriuretic peptide (NT-proBNP), and cardiac troponin-I (cTnI) in dogs with MVD has not been studied. We investigated plasma NT-proANP levels by MVD severity, and compared their clinical utility for assessing MVD severity. Clinically healthy dogs and client-owned dogs with MVD were examined. The diseased dogs were divided into three groups based on the American College of Veterinary Internal Medicine guidelines. Concentrations of plasma ANP, NT-proANP, NT-proBNP, and cTnI were analyzed by a commercial laboratory. Plasma ANP and NT-proANP concentrations increased significantly in stage B2 and C compared with that in the controls. They also increased significantly in stage C compared with that in stage B1. Plasma NT-proBNP and cTnI concentrations increased significantly in stage C compared with that in the controls and stage B1. Plasma NT-proANP concentration showed a strong positive correlation with ANP and NT-proBNP, and a weak positive correlation with E wave velocity, left atrial-to-aortic diameter ratio (LA/Ao raito), the left ventricular end-diastolic internal diameter corrected for body weight (LVIDDN), and VHS. The areas under the receiver-operating characteristic curve of NT-proANP used to identify dogs with each stage of MVD were comparable for other biomarkers. Circulating NT-proANP increase from the early stages of MVD, and it may be useful for assessing asymptomatic MVD. It can support echocardiographic data, but cannot be used alone to diagnose MVD.