Heptathela spiders of Okinawajima Is. were taxonomically revised using DNA sequence data. An analysis of mt-COI gene sequences revealed two clades (HY and HH), one of which (HY) was identified as Heptathela yanbaruensis because HY included specimens from Yona, the type locality of this species. To evaluate gene flow between HY and HH, the sequences of the nuclear 28S-rRNA and histone H3 genes were analyzed for specimens collected from two sympatric localities. In the two nuclear genes of HY and HH, all specimens had 5 homozygous positions with bases that differed between HY and HH. This indicates that there has been no gene flow between HY and HH, which is solid evidence for their reproductive isolation. Thus, it was concluded that HY and HH should be recognized as independent species, and HH is described as a new species under the name Heptathela helios n. sp. There were no good morphological diagnostic features other than the body size of the adult males of the two species (yanbaruensis ＜ helios); thus, females of these two species can be distinguished only by DNA sequence data.