Abstract: Rehmannia, a genus of Scrophulariaceae, is widely distributed in central, eastern and northern China. However, it is relatively difficult to distinguish species by traditional morphological methods due to rapid speciation of the genus. Recently developed DNA barcoding provides a new approach to identify species quickly and accurately. In this study, three candidate DNA noncoding regions (trnL-trnF, trnM-trnV and trnS-trnG) from the chloroplast genome and internal transcribed spacer (ITS) region from the nuclear genome were evaluated among 75 individuals of five species of Rehmannia, and identification efficiency was assessed using PWG-distance and Tree-Building methods. Results indicated that the five Rehmannia species could not be accurately distinguished by single or the combination of chloroplast DNA fragments with low species discrimination rates (0%-20%), but could by the combination of chloroplasts and nuclear DNA regions (trnS-trnG+ITS) with 100% discrimination rates. Thus, trnS-trnG+ITS can be considered as a potential barcode for species identification in Rehmannia. The combination of chloroplasts and nuclear fragments can be widely adopted for the discrimination of plant species when a single DNA barcode fails.