Ixiolirion is a genus of unresolved taxonomy. DNA barcoding is a technique that allows species identification using standardized DNA sequences. In this study, a total of 23 individuals, representing 2 Chinese Ixiolirion species, were sampled to test the effectiveness of 3 DNA barcodes [internal transcribed spacer (ITS), chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase] for species identification. Of the 3 DNA barcodes, ITS displayed the maximum level of polymerase chain reaction and sequencing success as well as the highest sequence variation. Intra-specific sequence distances of ITS, chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase were 0, 0, and 0-0.1%, respectively, with 8.3, 0.6, and 0.5% as mean inter-specific distances, respectively. All individuals of each species formed a monophyletic group (clade) in the neighbor-joining trees constructed using the 3 single-DNA barcodes. Our results demonstrated that ITS, chloroplast tRNA intron, and megakaryocyte-associated tyrosine kinase DNA markers could be used to identify Ixiolirion species. Our results indicate that DNA barcoding provides a reliable and effective means for discriminating Ixiolirion species, and is a robust tool for resolving taxonomic controversies of Ixiolirion in combination with morphology-based taxonomy.