Oyster diseases caused by pathogenic vibrios pose a major challenge to the sustainability of oyster farming. In France since 2012 a disease affecting specifically adult oysters has been associated with the presence of Vibrio aestuarianus. Here, by combining genome comparison, phylogenetic analyses and high throughput infections of strains isolated before or during the recent outbreaks, we show that virulent strains cluster into two V. aestuarianus lineages independently of the sampling dates. The bacterial lethal dose was not different between strains isolated before or after 2012. Hence the emergence of a new highly virulent clonal strain is unlikely. Each lineage comprises nearly identical strains, the majority of them being virulent, suggesting that within these phylogenetically coherent virulent lineages a few strains has lost their pathogenicity. Comparative genomics allowed the identification of a single frameshift in a non-virulent strain. This mutation affects the varS gene that codes for a signal transduction histidine-protein kinase. Genetic analyses confirmed that varS is necessary for infection of oysters and for a secreted metalloprotease expression. For the first time in a Vibrio species we show here that VarS is a key factor of pathogenicity