Ensuring the authenticity of raw materials used as herbs is a key step prior to producing medicines. "Gusuibu" is a traditional Chinese medicine for the treatment of bone diseases. Drynaria roosii Nakaike is the botanical origin of "Gusuibu". However, many "Gusuibu" adulterants which are morphologically similar, have been widely used in China. It is important to develop DNA-based markers to efficiently distinguish authentic "Gusuibu" from adulterants. In this study, 21 chloroplast genomes from seven species including D. roosii and six "Gusuibu" adulterant species were sequenced. The chloroplast genomes of D. roosii, D. sinica Diels, D. bonii Christ, D. delavayi Christ, D. quercifolia (L.) J. Sm., D. propinqua (Wall.) J. Sm., and Pseudodrynaria coronans (Wall.) Ching were 154,181 bp, 151,711 bp, 151,542 bp, 151,709 bp, 151,570 bp, 152,442 bp, and 151,466 by in length, respectively. Phylogenetic analysis indicated that whole chloroplast genomes could be used to distinguish D. roosii from adulterants and between each adulterant. Comparing chloroplast genome sequences, 12 protein-coding genes and eight intergenic sequences with high divergence in chloroplast genomes were identified to exploit specific DNA barcodes and sequence characterized amplified region (SCAR) markers. One specific DNA barcode and three SCAR markers, which were available to distinguish D. roosii from adulterants, were developed and four primer pairs were designed. The primer pairs for amplification of DNA barcode and SCAR markers designed in this study will be useful for economically and effectively distinguishing D. roosii from adulterants, and for guaranteeing the quality, safety, and effectiveness of "Gusuibu" herbs.