One of the most widely European farmed mollusc, the mussel Mytilus sp., has been subjected to massive mortalities located in Charente-Maritime (France) in spring 2014. The national surveillance network for mollusc health has reported a systematic detection of V. splendidus in all dying batches. V. splendidus is the type species of a clade composed of almost 20 known strains with variable pathogenicity on bivalves. In our study, we first developed a Multi Locus Sequence Analysis (MLSA), specific to V. splendidus group, based on fragments of 5 housekeeping genes: atpA, ftsZ, mreB, rpoD and topA. This tool was validated on reference strains and compared with individual gene analyses. It allowed a useful and reliable classification of V. splendidus closely-related strains. Thanks to MLSA, we then tried to classify genetically 23 strains isolated from healthy or dying mussels. 21 were classified within the Splendidus clade: in splendidus cluster (38%), in tasmaniensis cluster (24%), in artabrorum cluster (24%) and on distinct branches (14%). All of them were tested by injection to healthy adult mussels to identify possible pathogenic strains. Experimental trials revealed the presence of a strain called M3H, allied with the splendidus cluster and able to induce mortality in mussels with rates up to 80%. The M3H virulence was demonstrated by the recovery of the injected strain in dying animals, resulting from repeated experimental infections. Further work should be now conducted to explore the pathogenicity of the M3H strain towards different mussel batches and under various conditions.