A taxonomic study of the littoral earthworm genus Pontodrilus Perrier, 1874 was critically conducted based on material collected from marine sandy coasts along the east and west sides of the Thai-Malay Peninsula (Thailand and Malaysia) and selected localities in Japan. Comparative material from other parts of Southeast Asia, including Myanmar, Vietnam, Malaysia, Singapore and Indonesia, were also collected. Morphological examination identified almost all of the specimens as Pontodrilus litoralis, but as two morphotypes with some character differences in the diverticulum between them. A combined morphological and molecular phylogenetic analyses supported one of these morphotypes as a new species, P. longissimus. Statistical inference on morphometric data revealed significantly different size variations in the body length and diameter among the 14 populations of P. litoralis from Southeast Asia and Japan. One-way analysis of variance revealed significant difference in the body length and diameter among specimens from the different geographical sites. However, this distinction was not congruent with the phylogenetic relationship based on mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene sequence analysis. In addition, molecular analysis of 136 samples of P. longissimus using the 658 bp DNA fragment of COI revealed that the most frequent haplotype was present in four populations from the Andaman Sea, with a greater degree of genetic variation than in the Gulf of Thailand (GOT) clade. Bayesian inference, maximum likelihood and haplotype network analyses clearly showed that there were two geographically isolated populations of the P. longissimus lineages, one restricted to the GOT and the other to the Andaman Sea. The divergence and distribution of the worms are probably restricted by tidal circulation patterns around the peninsula and vicinities.