Abstract DNA barcoding is a useful tool to define operational taxonomic units based on standardized DNA regions. In this study, three chloroplast markers (rbcL, trnH-psbA and matK) and one nrDNA marker (ITS) were tested for species identification in Roscoea. The ITS and trnH-psbA regions showed high success rate of PCR amplification and bidirectional sequencing, as well as perfect discriminatory ability. On the contrary, rbcL possessed no genetic variation and matK was relatively difficult in PCR amplification and DNA sequencing. Combination of multiple markers greatly improved identification ability of DNA barcoding. ITS + trnH-psbA could effectively discriminate 90% species based on method of the Neighbor-Joining (NJ) tree. Awful PCR amplification and DNA sequencing of matK restricted its efficiency, although it showed rich genetic variability in Roscoea. Moreover, it might be more appropriate to treat Roscoea cautleoides var. pubescens as an independent species based on molecular data, namely R. pubescens Z. Y. Zhu.