The aim of this study was to evaluate the utility of a real-time PCR platform to estimate the DNA G+C content (mol%) and DNA–DNA hybridization (DDH) values in the genus Vibrio. In total, nine vibrio strains were used to determine the relationship between genomic DNA G+C content and T m (°C). The T m and HPLC datasets fit a linear regression curve with a significant correlation coefficient, corroborating that this methodology has a high correlation with the standard methodology based on HPLC (R2 = 0.94). Analysis of 31 pairs of vibrios provided a wide range of ΔT m values, varying between 0.72 and 12.5 °C. Pairs corresponding to strains of the same species or strains from sister species showed the lowest ΔT m values. For instance, the ΔT m of the sister species Vibrio harveyi LMG 4044T and Vibrio campbellii LMG 11216T was 5.2 °C, whereas the ΔT m of Vibrio coralliilyticus LMG 20984T and Vibrio neptunius LMG 20536T was 8.75 °C. The mean ΔT m values corresponding to pairs of strains with DDH values lower than 60 % or higher than 80 % were, respectively, 8.29 and 2.21 °C (significant difference, P<0.01). The high correlation between DDH values obtained in previous studies and the ΔT m values (R2 = 0.7344) indicates that the fluorimetric methodology is a reliable alternative for the estimation of both DNA G+C content and ΔT m in vibrios. We suggest that strains of the same Vibrio species will have less than 4 °C ΔT m. The use of a real-time PCR platform represents a valuable alternative for the development of the taxonomy of vibrios.