BackgroundCardiac biomarkers provide objective data that augments clinical assessment of heart disease (HD).Hypothesis/ObjectivesDetermine the utility of plasma N‐terminal pro‐brain natriuretic peptide concentration [NT‐proBNP] measured by a 2nd generation canine ELISA assay to discriminate cardiac from noncardiac respiratory distress and evaluate HD severity.AnimalsClient‐owned dogs (n = 291).MethodsMulticenter, cross‐sectional, prospective investigation. Medical history, physical examination, echocardiography, and thoracic radiography classified 113 asymptomatic dogs (group 1, n = 39 without HD; group 2, n = 74 with HD), and 178 with respiratory distress (group 3, n = 104 respiratory disease, either with or without concurrent HD; group 4, n = 74 with congestive heart failure [CHF]). HD severity was graded using International Small Animal Cardiac Health Council (ISACHC) and ACVIM Consensus (ACVIM‐HD) schemes without knowledge of [NT‐proBNP] results. Receiver‐operating characteristic curve analysis assessed the capacity of [NT‐proBNP] to discriminate between dogs with cardiac and noncardiac respiratory distress. Multivariate general linear models containing key clinical variables tested associations between [NT‐proBNP] and HD severity.ResultsPlasma [NT‐proBNP] (median; IQR) was higher in CHF dogs (5,110; 2,769–8,466 pmol/L) compared to those with noncardiac respiratory distress (1,287; 672–2,704 pmol/L; P < .0001). A cut‐off >2,447 pmol/L discriminated CHF from noncardiac respiratory distress (81.1% sensitivity; 73.1% specificity; area under curve, 0.84). A multivariate model comprising left atrial to aortic ratio, heart rate, left ventricular diameter, end‐systole, and ACVIM‐HD scheme most accurately associated average plasma [NT‐proBNP] with HD severity.Conclusions and Clinical ImportancePlasma [NT‐proBNP] was useful for discriminating CHF from noncardiac respiratory distress. Average plasma [NT‐BNP] increased significantly as a function of HD severity using the ACVIM‐HD classification scheme.