The phylogenetic relationships among the species belonging to the family Myxinidae are still debatable. The mitochondrial DNA sequences from the large ribosomal RNA gene may be of great value for systematic and phylogenetic studies within families. Partial sequences of the 16S rRNA gene were obtained for comparisons among the following hagfish species, Paramyxine nelsoni, Paramyxine sheni, Paramyxine taiwanae, Paramyxine yangi, Paramyxine cheni, Eptatretus burgeri, Eptatretus stouii, Eptatretus cirrhatus, Myxine glutinosa, Myxine formosana, Myxine circifrons, Myxine sp1, and Myxine sp2. The boundary of four Paramyxine species (P. sheni, P. taiwanae, P. nelsoni, and P. yangi) from 16S rRNA sequences is ambiguous, however, they are valid based on our unpublished isozyme data as well as the gill aperture arrangement pattern. Both NJ and MP trees constructed from the present molecular data indicate that the genus Paramyxine is diphyletic and Eptatretus paraphyletic. The complexity of Eptatretus and Paramyxine in the clade would not be solved until the farther departed P. cheni is included to form a new clade under the genus Eptatretus. The other clade of Myxininae contains but single genus Myxine.