Aim To provide a rapid and reliable method for identifying the fork medicine Stellaria media (Linn.) Cyr. (Herba Stellariae mediae) (Caryophyllaceae) from its adulterant Myosoton aquati-cum (L.) Fries (Herba Myosoti aquatici) (Caryophyllaceae) by polymerase chain reaction (PCR) technology. Methods A molecular genetic approach has been developed to identify S.media for the first time. 5S-rRNA spacer domain was amplified by PCR from the isolated genomic DNA, and the PCR pro-ducts were then sequenced. Results The nucleotide sequences of S.media and M.aquaticum were measured to determine their identity. Furthermore, the nucleotide sequences of three Stellaria species, S.vestita, S. longifolia and S.radians, were also measured for the sake of providing the evidence of the biological phylogeny of Stellaria. Diversity between DNA sequence and restriction enzyme mapping among a variety of the species was found in their 5S-rRNA spacer domains. Conclusion The 5S-rRNA spacer domains can be used as a molecular marker for differentiating S.media from M.aquaticum and in phylogenetic studies of Stellaria.