Abstract: DNA barcoding refers to the application of a small number of DNA fragments to achieve reliable, automatable species-level identification. In this study, the suitability of four candidate sequence regions were assessed-mitochondrial COI-5P and cox2-3 spacer, plastid rbcL and UPA-for species delimitation and discrimination in family Batrachospermaceae. The percentage of successful PCR amplifications of COI-5P, cox2-3 spacer, UPA, and rbcL markers was 96%, 100%, 96%, and 98%, respectively. COI-5P, UPA, and cox2-3 spacer sequence lengths were amenable to the acquisition of bidirectional sequencing reads using single primer pairs and met our size criterion of 300—800 bp. Phylogenetic analyses revealed that all four sequence regions were useful for species-level identification in the genus Batrachospermum except for some allied species. The two Chinese endemic species B. hongdongense and B. longipedicellatum were unable to differentiate from B. arcuatum using COI-5P, cox2-3 spacer, and rbcL markers, excepted for the UPA region. For species-level identification, the UPA locus exhibited the highest interspecific distances. We therefore recommended the plastid UPA gene as a standard DNA barcode in Batrachospermaceae, but acknowledge that there are no shared alleles between the endemic species.