Lysin is a 16kDa acrosomal protein used by abalone sperm to create a holein the egg vitelline envelope (VE). The interaction of lysin with the VE isspecies-selective and is one step in the multistep fertilization processthat restricts heterospecific (cross-species) fertilization. For thisreason, the evolution of lysin could play a role in establishing prezygoticreproductive isolation between species. Previously, we sequenced spermlysin cDNAs from seven California abalone species and showed that positiveDarwinian selection promotes their divergence. In this paper an additional13 lysin sequences are presented representing species from Japan, Taiwan,Australia, New Zealand, South Africa, and Europe. The total of 20 sequencesrepresents the most extensive analysis of a fertilization protein to date.The phylogenetic analysis divides the sequences into two major clades, onecomposed of species from the northern Pacific (California and Japan) andthe other composed of species from other parts of the world. Analysis ofnucleotide substitution demonstrates that positive selection is a generalprocess in the evolution of this fertilization protein. Analysis ofnucleotide and codon usage bias shows that neither parameter can accountfor the robust data supporting positive selection. The selection pressureresponsible for the positive selection on lysin remains unknown.