Sea urchins of the genus Echinometra are abundant on Okinawa reef flats in southern Japan. The Okinawan Echinometra is subdivided into four sympatric and closely related species (Ea, Eb, Ec, and Ed). To elucidate the relationships between them, we have examined the effect of egg jelly on the acrosome reaction, a process sea urchin spermatozoa undergo before fertilization. When Echinometra spermatozoa were treated with eggjelly, the acrosome reaction in each species was, as expected, effectively induced by jelly from the same species. The acrosome reaction in Ed spermatozoa was induced by egg jelly regardless of species, whereas the acrosome reaction of Eb spermatozoa took place only when it was treated with Eb egg jelly. The acrosome reaction of Ea spermatozoa was induced by jelly from species Ea or Eb, while Ec spermatozoa responded to egg jelly from Ea, Ec, or Ed. These results suggest that the separation of these four species of Okinawan Echinometra has taken place quite recently. An additional finding of our study is that the rate of sperm motility differs from species to species. In order from slowest to fastest, the species are Ed, Ec, Eb, and Ea. Motility may be related to reproductive isolation among the four species of Echinometra.