The Vibrio splendidus clade is the biggest in Vibrionales composed of 11 described species (25). Diversification of these species may have occurred 260 million years ago. The main driving forces of speciation in this clade have never been studied. Population biological parameters (population base recombination rate (ρ), population base mutation rate (θ), and index of association (Ia)) were determined among 16 strains of 9 defined species in the Splendidus cluster. A comparison of individual gene phylogeny indicated significant incongruence in tree topology, which suggests the occurrence of recombination between species. Homologous recombination between species was detected at four loci. However, the mutation rate θ was higher than the recombination rate ρ, suggesting that mutation is the main driving force in the diversification of V. splendidus-related species.