Seafood has been identified as an important source of Vibrio cholerae in Thailand, especially in the Southern coastal region. In this study, we isolated and characterized V. cholerae from seafood obtained from several markets in Hat Yai city, Southern Thailand. A total of 100 V. cholerae isolates were obtained from 55 of 125 seafood samples. The dominant serotype was non-O1/non-O139. Polymerase chain reaction (PCR) analysis was used to detect the presence of pathogenesis-related genes. The stn/sto and hlyA El Tor virulence genes were detected in 20% and 96% of the isolates, respectively. None of the isolates were positive for the ctxA, tcpA, zot, and ace genes. Only 6% of the isolates carried the T3SS gene (vcsV2); however, the majority of the isolates (96%) carried the T6SS gene (vasH). Representative isolates (n=35) that exhibited various virulence gene patterns were randomly selected and analyzed for their hemolytic activity, antibiotic susceptibility, biofilm formation, and genotype. Hemolytic activity using sheep red blood cells was detected in only one of the hlyA-negative isolates. Apart from ampicillin, all isolates were pansusceptible to five test antibiotics. Biofilm production was observed in most of the isolates, and there was no difference in the presence of a biofilm between the smooth and rugose isolates. Using the enterobacterial repetitive intergenic consensus-PCR method, clonal relationships were observed among the isolates that exhibited identical virulence gene patterns.