AbstractConopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O‐superfamily conotoxins was found in a worm‐hunting cone snailConus miles. Five novel cDNA sequences encoding O‐superfamily precursor peptides were identified inC. milesnative to Hainan by RT‐PCR and 3′‐RACE. They share the common cysteine pattern of the O‐superfamily conotoxin (CCCCCC, with three disulfide bridges). The predicted peptides consist of 27–33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences fromC. milesand the otherConusspecies. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter‐ and intra‐species. Despite the O‐superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types ofConusand discovered in severalConusspecies, it was for the first time that the newly found five O‐superfamily peptides in this research came from the vermivorousC. miles. So far, conotoxins of the O‐superfamily whose properties have been characterized are from piscivorous and molluscivorousConusspecies, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function. Copyright © 2006 European Peptid