Abstract: Viola philippica is a widely used herb in Chinese traditional medicine. However, V. mandshurica is found as one of common substitutes or adulterants of V. philippica in the commercial market. In order to authenticate the two herbal species, an improved randomly amplified polymorphic DNA (RAPD) and Sequence-Characterized Amplified Region (SCAR) were performed to obtain species-specific DNA fragments. Three 10-mer random primers SBS-A8, SBS-I10 and SBS-Q6 were used for screening of specific RAPD markers and the former two primers demonstrated different amplification band patterns. Two candidate specific bands showed in two taxa by SBS-A8 primer were successfully cloned and sequenced respectively. Based on the RAPD marker sequences, four pairs of SCAR primers from V. philippica (VPF1/VPR1 and VPF2/VPR2) and V. mandshurica (VMF1/VMR1 and VMF2/VMR2) were designed. VPF1/VPR1 and VPF2/VPR2 primers yielded expected 694 bp and 403 bp amplicons with the V. philippica DNA only. DNA amplification using VMF1/VMR1 primers generated a single 574 bp band only in V. mandshurica, while VMF2/VMR2 primers produced 261 bp bands in two species. The results suggested that RAPD and SCAR analysis are effective in distinguishing genuine V. philippica. The methodology is a valuable tool to authenticate other morphologically similar herbal medicinal materials.