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Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4[ratio ]K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3[ratio ]K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3[ratio ]K6 and O4[ratio ]K68 strains and for comparison, non-O3[ratio ]K6 and non-O4[ratio ]K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3[ratio ]K6 and O4[ratio ]K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3[ratio ]K6 and O4[ratio ]K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3[ratio ]K6 and O4[ratio ]K68 strains formed a cluster with 78–91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3[ratio ]K6 and non-O4[ratio ]K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.