The 16S rDNA - RFLP types for six Vibrio species (V. fluvialis, V. proteolyticus, V. vulnificus, V. mimicus) including two core group members, V. alginolyticus and V. parahaemolyticu s, and Grimontia (Vibrio) hollisae were determined using PCR-RFLP analysis. Six tetrameric restriction enzymes (Alu I, Cfo I, Dde I, Hae III, Msp I, and Rsa I) were selected for RFLP analysis. V. alginolyticus, V. parahaemolyticus, and V. proteolyticus showed the same RFLP pattern following digestion with four of the six used restriction enzymes: CfoI, DdeI, MspI, and RsaI. Various restriction enzyme combinations generated digests recognizable as distinct RFLP types for each of the assayed Vibrio species. In particular, AluI single digestion produced species specific band patterns that enabled the differentiation between these Vibrio species. Dendrogram based on restriction patterns showed that two Vibrio core group members, V. alginolyticus and V. parahaemolyticus were closely related having a similarity over 90%. Although the observed RFLP pattern for Grimontia hollisae shared several common bands with other Vibrio spp., G. hollisae results were still clearly distinct from Vibrio spp. RFLP types for all restriction enzymes tested. If restriction enzymes are aptly selected, PCR-RFLP analysis is still a rapid and effective tool for differentiating Vibrio species.