Vibriosis caused by Vibrio vulnificus is a major problem in aquatic animals, particularly brown marble groupers (Epinephelus fuscoguttatus). V. vulnificus biotype I has recently been isolated and classified into subgroups SUKU_G1, SUKU_G2, and SUKU_G3 according to the different types of virulence genes. In a previous study, we have shown that biotype I V. vulnificus strains were classified into three subgroups according to the different types of virulence genes, which exhibited different phenotypes in terms of growth rate and virulence. To gain insight into the different genetic features revealed by the potential virulence mechanisms of V. vulnificus in relation to a spectrum of pathogenesis, comparative genomic analyses of three biotype I V. vulnificus strains belonging to different subgroups (SUKU_G1, SUKU_G2, and SUKU_G3) were performed. The V. vulnificus genome is composed of two circular chromosomes with average sizes of 3 Mbp and 1.7 Mbp that are evolutionarily related based on the analysis of orthologous genes. A comparative genome analysis of V. vulnificus revealed 5200 coding sequences, of which 3887 represented the core genome and the remaining 1313 constituted the dispensable genome. The most virulent isolate (SUKU_G1) carries unique enzymes that are important for lipopolysaccharide (LPS) and capsular polysaccharide (CPS) synthesis, as well as flagellar glycosylation, and harbors another type of repeat in toxin (RTX) and bacterial defense mechanisms. The less virulent isolate (SUKU_G2) shares enzymes related to CPS biosynthesis or flagellar glycosylation, while the avirulent isolate (SUKU_G3) and a less virulent isolate (SUKU_G2) share enzymes related to the production of rare sugars. Interestingly, the isolates from the three subgroups containing specific CMP-N-acetylneuraminate-producing enzymes that are correlated with their growth abilities. Collectively, these observations provide an understanding of the molecular mechanisms underlying disease pathogenesis and support the development of strategies for bacterial disease prevention and control.