Members of the freshwater halfbeak genus Dermogenys are hard to identify to the species level, despite several previous attempts to isolate fixed meristic, morphometric and colour pattern differences. This has led to ongoing confusion in scientific literature, records of species occurrence, and entries in museum collections. Here, a DNA barcoding study was conducted on the genus to gain further understanding of its taxonomic status across the Southeast Asian region. Fish were collected from 33 localities, spanning freshwater and brackish habitats in Malaysia, Western Indonesia, Thailand and Vietnam. In total, 290 samples of Dermogenys spp. were amplified for a 651 base pair fragment of the mitochondrial cytochrome oxidase c subunit I (COI) gene. Analysis was able to successfully differentiate the three species: D. collettei, D. siamensis, D. sumatrana; reveal the presence of a new putative species, Dermogenys sp., that was sampled in sympatry with D. collettei at three locations; as well as uncovering two genetic lineages of a fifth species, D. bispina, that display non-overlapping geographical distributions in drainages of northern Borneo; Kudat and Sandakan. This study expands the barcode library for Zenarchopteridae, demonstrates the efficacy of DNA barcoding techniques for differentiating Dermogenys species, and the potential thereof in species discovery.