Although seagrass species in the genus Halophila are generally distributed in tropical or subtropical regions, H. nipponica has been reported to occur in temperate coastal waters of the northwestern Pacific. Because H. nipponica occurs only in the warm temperate areas influenced by the Kuroshio Current and shows a tropical seasonal growth pattern, such as severely restricted growth in low water temperatures, it was hypothesized that this temperate Halophila species diverged from tropical species in the relatively recent evolutionary past. We used a phylogenetic analysis of internal transcribed spacer (ITS) regions to examine the genetic variability and evolutionary trend of H. nipponica. ITS sequences of H. nipponica from various locations in Korea and Japan were identical or showed very low sequence divergence (less than 3-base pair, bp, difference), confirming that H. nipponica from Japan and Korea are the same species. Halophila species in the section Halophila, which have simple phyllotaxy (a pair of petiolate leaves at the rhizome node), were separated into five well-supported clades by maximum parsimony analysis. H. nipponica grouped with H. okinawensis and H. gaudichaudii from the subtropical regions in the same clade, the latter two species having quite low ITS sequence divergence from H. nipponica (7-15-bp). H. nipponica in Clade I diverged 2.95 ± 1.08 million years ago from species in Clade II, which includes H. ovalis. According to geographical distribution and genetic similarity, H. nipponica appears to have diverged from a tropical species like H. ovalis and adapted to warm temperate environments. The results of divergence time estimates suggest that the temperate H. nipponica is an older species than the subtropical H. okinawensis and H. gaudichaudii and they may have different evolutionary histories.