V. vulnificus is a heterogeneous bacterial species that comprises virulent and avirulent strains from environmental and clinical sources that have been grouped into three biotypes. To validate the typing methods proposed to distinguish clinical from environmental isolates, we performed phenotypic (API 20E, API20NE and BIOLOG tests) and genetic (ribotyping and DNA polymorphisms at several loci) studies with a large strain collection representing different biotypes, origins and host range. No phenotypic method was useful for biotyping or grouping strains with regard to origin of the isolate and only BIOLOG system was reliable to identify the strains at species level. The DNA polymorphisms divided the population into three major profiles: profile 1 were vcg type C, 16S rRNA type B and vvh type 1 and included most of biotype 1 human septicemic isolates; profile 2 were vcg type E, 16S rRNA type A and vvh type 2 and included all biotype 2 isolates together with biotype 1 isolates from fish, water and some human isolates; and the last one, profile 3, were vcg type E, 16S rRNA type AB and vvh type 2 and composed by biotype 3 strains. Ribotyping divided the species into two groups, one that included profile 1 isolates of biotype 1, and the other including isolates of all three biotypes belonging to the three profiles described. In conclusion, no genotyping system was able to distinguish neither clinical from environmental strains nor biogroups within V. vulnificus species, which suggests that new typing methodologies useful for Public Health have to be developed for this species.