Abstract: OBJECTIVE To choose the most suitable extraction method of total DNA in medicinal Fritillaria L.. METHODS Using the kit method, modified CTAB method, modified SDS method total DNA of Fritillaria L. were extracted; the potency and purity of the total DNA were detected by nucleic acid protein detector and agarose gel electrophoresis method; ISSR-PCR amplification detection obtained DNA quality. RESULTS higher yield of genomic DNA can be extracted from the plant Fritillaria L. by 3 kinds of methods. But compared with the total DNA, kit method was the highest, best quality of the extracted, it could amplify the bands rich and clear, and high stability, simple operation and short time. The purity and quality of total DNA extracted by modified CTAB method or SDS method were inferior those by kit method. Moreover, CTAB method or SDS method was complex and time-consuming operation, not for downstream molecular biology experiments. CONCLUSION The kit method is the best way to extract total DNA from medicinal plants of Fritillaria L., and can be used directly in PCR and other biological research.