Nassarius causes many poisonings in China and other Asian countries. The toxicity of Nassarius is related to species and populations. However, the molecular assignment of different toxic Nassarius samples is still unclear, which deteriorates the poisoning. Our study combined toxicity, toxin detection and multiple genes DNA barcoding to assign the various toxic Nassarius species and populations from China coast. The toxicity test indicated that different Nassarius species and different populations within one Nassarius species had different toxicity. Based on High Performance Liquid Chromatography, the toxin was confirmed to be TTX. The toxin content varied in different samples. The COI, 16S and ITS sequences revealed different toxic Nassarius species as monophyletic clades. All the samples tested have been assigned to species level. However, the different toxic populations within N. succinctus, N. variciferus and N. semiplicata did not form separated clades, and the toxic and nontoxic species did not form two clades separately. Our study constructed the relationship between toxicity and taxonomy assignment of different toxic Nassarius samples based on toxicity detection and multiple gene DNA barcoding, which provides the basis for controlling poisoning and studying the toxin-resistance difference in Mollusca. It is also indicated that the toxicity of Nassarius is really related with species at the genetic level, but not absolutely related with populations.