Abstract: In the present study, tetrodotoxin (TTX) extracted from eyes, skin, liver, intestine and gonads of Diodon hystrix were evaluated for toxicity study. The COX1 gene sequencing revealed 99% similarity to D. hystrix . This identification of D. hystrix from Chennai coastal region is the first molecular report providing new data about their geographical distribution. The GC-MS analysis revealed the presence of TTX toxin in all the crude extracts of the fish except eye. Human lymphocyte chromosome aberration assay was tested using crude extracts containing TTX (0.5 mg mL–1) which caused spindle fibre aberration and comet assay showed lack of DNA damage. In support of genotoxicity, all the crude extracts containing TTX was tested on zebra fish, using 1 mL of 5 mg mL–1 concentration stock solution. The results suggest that extracts from skin, gonads and liver of D. hystrix caused 100% mortality within 72 h. Based on these observations it is suggested that D. hystrix from chennai coastal region as food is a potential risk to humans.