In this article, the genotypes for Vibrio alginolyticus were analyzed based on collagenase gene by PCR-single strand conformation polymorphism (SSCP) method and their relationship to the virulence for marine fish were studied by a bacteria challenging experiment. Results showed total 13 genotypes designated as A1, A2, A3, A4, B, C, D, E, F, G1, G2, H, and I were identified. Among them, isolates from type A1 were dominant in abundance (48.1%) and the isolates from Nanyou had the highest polymorphisms, in which 11 genotypes were identified from 25 isolates. Moreover, the bacteria challenging experiment showed the virulence of V. alginolyticus was correlated to that of the collagenase-SSCP type (r = 0.438, P < 0.01), which demonstrated the strains from type A1, A2, A3, B, C, F, G1, and I were low-virulent isolates, while those from type A4, D, E, G2, and H were high virulence. These data will provide a new understanding of the relationship between virulence of V. alginolyticus and its molecular marker.