Fragments of the nuclear ribosome(ITS)and the chloroplast(matK and rbcL)regions were successfully suitable for phylogenetic relationship analysis.With sequencing by PCR amplification,the paper analyzed the phylogenetic relationship of 5 Zosteraceae species(Zostera marina,Z.japonica,Z.caespitosa,Z.asiatica and Phyllospadix iwatensis),which were collected from Ireland,Japan,Korea,China and GenBank.Partial nucleotide sequences of matK, rbcL gene and ITS were analyzed for five Zosteraceae species,and nucleotide composition analysis indicated a strong bias against cytimidine(C)in three fragments.172 nucleotide substitutions were found in ITS fragment,showing the high genetic polymorphism.52 nucleotide substitutions were checked in matK gene and 20 nucleotide substitutions were checked in rbcL gene,most of them were synonymous transitions at the third codon positions.The five species had a certain degree of differentiation at the amino acid level.Based on partial sequences of matK, rbcL and ITS gene,phylogenetic trees were constructed by NJ,MP,ML and Bayes methods and the results were consistent and the phylogenetic trees showed three separate lineages.The minimum pairwise divergences percentage in the samples of Z.japonica and other Zostera species was 19.33% and much higher than the standard(9.60%-28.80%)among genera of most angiosperm and it would be the generic rank from the molecular data.Based on the synonymous nucleotide substitution rate of the rice chloroplast genome matK combined with rbcL gene,we estimated the divergence time between 5 Zosteraceae species approximately arranged from Eocene to Pliocene.The ITS sequences of all Z.marina samples were identical,which fell into the same cluster.It was concluded that variation within ITS region of Z.marina was not correlated to their geographical distance,and ITS region was unsuitable for identification of population groups on a regional or oceanic scale.The paper further elaborated phylogenetic relationship of 5 Zosteraceae species,and also provided theoretic basis for seagrass phylogeny in China.