Based on the ITS2 and psbA-trnHsequences, molecular biological identification and genetic relationship of Fritillaria cirrhosa with its relative species were carried out. In this paper, the PCR-RFLP method specified by the Chinese Pharmacopoeia was performed on all samples at first. Secondly, the ITS2 and psbA-trnH sequences of all samples were amplified. Then, the amplified products were used to analyze the genetic distance, construct the phylogenetic tree, assess the identification efficiency, and evaluate the genetic relationship as well. The result showed that all the samples were divided into two groups by PCR-RFLP method. The samples in the first group, including Fritillaria ussuriensis, Fritillaria thunbergii and Fritillaria pallidiflora, could not be digested by SmaI, while the other samples in the second group, including Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa, could be digested by SmaI. Then, ITS2 and psbA-trnH sequences of all samples were obtained. The length of various ITS2 sequences were distributed from 235 to 239 bp, and the average intra- and inter-specific genetic distance were 0.001 and 0.022, respectively. NJ tree showed that all samples were separated into "Northern Fritillaria" group (Fritillaria ussuriensis and Fritillaria pallidiflora) and "Southern Fritillaria" group (Fritillaria thunbergii, Fritillaria mellea, Fritillaria sinica, Fritillaria cirrhosa var. ecirrhosa Franch, Fritillaria unibracteata var. longinectarea and Fritillaria cirrhosa). The latter group could be further divided into Fritillaria thunbergii and Fritillaria cirrhosa subgroup, and the species in Fritillaria cirrhosa subgroup had close phylogenetic relationships. The length of psbA-trnH sequences was distributed from 337 to 373 bp, and the intra- and inter-specific genetic distance were 0.263 and 0.329, respectively. The samples in this paper could not be clustered effectively by NJ tree. This indicated that the ITS2 sequences were not only able to identify Fritillaria cirrhosa with its partial relative species quickly and accurately, but also clarify the relationship between different Fritillaria species. Therefore, it provided an important theoretical foundation for the development of molecular markers, effective protection, and rational development and utilization of Fritillaria resources.