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 (R(2) = 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 4044(T) and Vibrio campbellii LMG 11216(T) was 5.2 °C, whereas the ΔT(m) of Vibrio coralliilyticus LMG 20984(T) and Vibrio neptunius LMG 20536(T) 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 (R(2) = 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.