Vibrio is a major sea food pathogen which needs to be monitored regularly for devising appropriate control strategy to ensure food safety among sea food consumers and handlers. The present study aims to ascertain the occurrence of Vibrio spp. in seafood viz fishes, prawn/shrimps, crabs and water samples collected from various fish markets in and around Chennai city, Tamil Nadu, India. A total of 175 samples were screened, of which 106 (60.57%) samples were found positive by PCR targeting the 16s rRNA of Vibrio genus and all the positive isolates were subjected to toxR gene identification by Multiplex PCR for species level identification. At the species level 28(26.41%) were positive for Vibrio parahemolyticus (Vp), 6 (5.67%) positive for Vibrio Cholerae (Vc), 2 (1.89%) positive for Vibrio vulnificus (Vv). The presence of mixed vibrio species were also observed of which 56 samples (43.40%) revealed the presence of Vp and Vc, 7 (6.60%) with Vp and Vv, 3 (2.84%) with Vv and Vc and 14 samples with (13.20%) Vp, Vv and Vv. The present study identified the presence along with mixed Vibrio spp. in most of the samples tested from the study area.