Abstract: Seagrasses are composed of four families belonging to angiosperms and they are thought to become adaptive to aquatic life independently. The marine monocotyledonous Zosteraceae are one such family, which place in the order of Helobiae using traditional classifications and are typically circumscribed to comprise three genera (Heterozostera, Phyllospadix and Zostera) with approximately 18 species. Since Zosteraceae had been reported, the studies about the family were mainly focused on their morphological characters, physiological and biochemical characters and their flora. But the phylogenetic relationship between taxa of Zosteraceae was still hard to guarantee in terms of similar morphological characteristics. With sequencing by PCR amplification, we reconstructed the phylogenetic relationship of Zosteraceae based on fragments of the chloroplast (matK) and the nuclear ribosome (ITS) regions, which were successfully suitable for phylogenetic relationship analysis. Four species were collected from China and Korea, and eleven species were obtained from GenBank. The nucleotide composition analysis indicated a strong bias against cytimidine (C) in both fragments. 228 nucleotide substitutions were found in ITS gene, showing the high genetic polymorphism. 249 nucleotide substitutions were checked in matK gene, and most of them were synonymous transitions at the third codon positions. The Zosteraceae species had a certain degree of differentiation at the amino acid level. Based on partial sequences of matK and ITS gene, phylogenetic trees were constructed by neighbor joining, maximum parsimony, maximum likelihood and bayesian inference methods and the four methods produced trees with largely congruent topology. These phylogenetic trees showed four separate lineages: (1) subgenus Zostera, (2) Heterozostera, (3) subgenus Zosterella and (4) Phyllospadix. The pairwise percentage divergence values in the samples of subgenus Zostera and subgenus Zosterella were from 29.09% to 35.51% and were much higher than the standard values (9.60% to 28.80%) among genera of most angiosperm. The results suggested genetic differentiation of these two subgenus had reached the level of generic variation. Our results lend support to the classification by Tomlinson and Posluszny, who recommended taxonomic delimition of Zosteraceae as four genera: Heterozostera (one sp.), Nanozostera (eight spp.), Phyllospadix (five spp.) and Zostera (four spp.). The molecular evidence suggested that Phyllospadix was the most divergent taxon, while Heterozostera and Nanozostera were the most closely related taxa. The phylogenetic relationship was more complex, so, further molecular and morphological investigations were needed to classify the phylogentic relationships of Zosteraceae.