Abstract Plasma NT-proBNP has previously been evaluated in dogs with degenerative mitral valve disease (DMVD). However, reference intervals (RI) established according to the Clinical Laboratory and Standards Institute (CLSI) recommendations have never been provided. The objectives of this prospective study were to assess effects of breed, body weight, age, and sex on plasma NT-proBNP, and to establish RI according to CLSI for this biomarker in a large population of dogs predisposed to DMVD. 183 Healthy small-sized dogs from 7 breeds were included. Assays were performed by ELISA. Effects of covariates were tested using a general linear model. Although a sex effect was demonstrated (P=0.01), no significant effect of breed, body weight or age was shown. The proposed RI was 157-2842pmol/L. 7% of dogs had plasma NT-proBNP >2617pmol/L, and were considered as outliers despite normal cardiovascular examination. In conclusion, plasma NT-proBNP may be high in a few healthy small-sized dogs.