Objective To differentiate the sequences of Cyt b gene Agkistrodon from its adulterants sold in the market in order to provide molecular evidence for identification of Agkistrodon. Methods Cyt b gene was used to sequence and analyze Agkistrodon and its adulterants sold in the market. Results There was a mixed phenomenon in Agkistrodon species. The differences of Cyt b gene sequence between Agkistrodon and its adulterants sold in the market are significant: the difference rates among the species of Agkistrodon are 0%—0.91%, the difference rates between Agkistrodon and its adulterants are 18.57%—23.78%. Conclusion The characteristics of Cyt b gene sequence can be used as a better molecular marker for authenticating Agkistrodon from its adulterants.