Abstract: Penthorum sedoides L. and Penthorum chinense Push is closely related aneuploids in genus Penthorum L. based on the previous data. However, P. chinense are treated as variety or subspecies of P. sedoides successively by botanists and little was known about their relationships on molecular level. An improved randomly amplified polymorphic DNA (RAPD) and Sequence Characterized Amplified Region (SCAR) were performed to obtain their genetic characterizations and species-specific DNA fragments. Obviously, five 10-mer random primers SBS-A3, SBS-I3, SBS-I18, SBS-M6 and SBS-Q9 demonstrated different fingerprints. Six candidate specific bands (I3-PS, I3-PC, I18-PC, M6-PS, Q9-PS and Q9-PC) displayed in two taxa were successfully cloned and sequenced. Based on these sequences, six pairs of SCAR primers from P. sedoides (I3PSF/I3PSR, M6PSF/M6PSR and Q9PSF/Q9PSR) and P. chinense (I3PCF/I3PCR, I18PCF/I18PCR and Q9PCF/Q9PCR) were designed, respectively. However, four of six primer pairs yielded amplicons common in the two taxa. The DNA amplification using Q9PSF/Q9PSR primers generated a single 555 bp band only in P. sedoides and Q9PCF/Q9PCR primers produced a 760 bp fragment unique to P. chinense. The results suggested that P. sedoides and P. chinense are still closely related, although there are a lot of variations in RAPD genetic sites. The developed RAPD and SCAR techniques are effective and useful in revealing genetic characterizations of P. sedoides and P. chinense. Also, the SCAR analysis based on the improved RAPD method is powerful in authentication of species with close relationships.