Vibrio alginolyticus is a serious bacterial pathogen that hampered the whole industry of fish farming in Guangdong, China. In order to facilitate epidemiologic studies and improve the control of disease, we developed a highly efficient method of genotyping for V. alginolyticus using infrequent-restriction-site PCR (IRS-PCR) technology. With the enzyme combination of NotI-HhaI, optimized, unique, and easy-to-interpret patterns were generated. Forty-five V. alginolyticus isolates from aquatic animals and the marine environment in Daya Bay in Guangdong were fingerprinted by IRS-PCR and pulsed-field gel electrophoresis (PFGE). The reproducibility of the IRS-PCR method was high (100%) in conformity with PFGE. When primer PN-T or PN-G was used, IRS-PCR obtained the identical clustering as PFGE, producing 24 different patterns, respectively. Moreover, IRS-PCR presented a high discriminatory power (D=0.953) identical to that of PFGE. In conclusion, the IRS-PCR fingerprinting method using NotI-HhaI can provide a potentially useful tool for epidemiologic investigations of V. alginolyticus isolates.