A fluorescent visual identification system of Paris polyphylla var. yunnanensis was established based on internal transcribed spacer barcoding. It is proposed for the first time that P. polyphylla var. yunnanensis should be divided into two types of genotypes: YN-I and YN-II according to single nucleotide polymorphism of internal transcribed spacer. In order to avoid false-negative results, two pairs of specific primers for YN-I and YN-II were designed, respectively, and specific visual fluorescent identification systems was established by SYBR Green I fluorescent dye was directly introduced into the PCR system which can be observed directly with the naked eye for the green fluorescent color of PCR system. Therefrom, it has realized the rapid and directly visual identification of two genotypes of P. polyphylla var. yunnanensis from its common nine adulterants. This study proposed for the first time the existence of different genotypes on the legal basis of Rhizoma Paridis, and provided a model for the accurate identification of different genotypes.