To optimize indices of molecular identification for authentication of Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix, four indices, including sequence similarity, specific positions, genetic distance and phylogenetic tree, were compared based on trnL-trnF sequences. Total DNA was extracted from Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix, and trL-trnF sequences were amplified and sequenced. Sequence similarity was calculated by BLAST analysis. Specific positions were compared by DNAman software. Genetic distance and phylogenetic tree were analyzed by Mega software. The results showed that the inter-specific and intra-specific similarity of P. ginseng and P. quinquefolius respectively was 100% and 99. 6%. There were four specific positions at G153A, T463A, C732G and T818C. The inter-specific genetic distance (0) of trL-trnF sequences was lower than intra-specific genetic distance (0. 004). P. ginseng can be distinguished from P. quinquefolius based on the phylogenetic tree. It is concluded that Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix can be authenticated by identification indices of sequence similarity, specific positions, genetic distance and phylogenetic tree. Index of specific positions based on trnL-trnF sequences is the most efficient index to authenticate Ginseng Radix et Rhizoma and Panacis Quinquefolii Radix.