Larval fish ecology is poorly characterized because sampling is difficult and tools for phenotypically identifying larvae are poorly developed. While DNA barcoding can help address the latter problem, ‘universal’ primers do not work for all fish species. The Roanoke River in the southeastern United States includes seven darters (Family Percide: Tribe Etheostomatini). We made 393 collections of larval fishes in 2015 and 2018, examined darter larvae for morphometric and pigmentation traits, developed PCR primers amplifying darter DNA, and evaluated three gear types for collecting larval darters. Amplified DNA sequences for 1351 larvae matched archived mitochondrial cytochrome oxidase I sequences for darters occurring in the ecosystem. Larval darters were classified to genus with 100% accuracy using the ratio of pectoral fin length to body length; however, identification to species using morphometrics alone was subject to a misclassification rate of 11.8%, which can be resolved by considering pigmentation patterns. Gear-types varied considerably in their capture efficacy for larval darters; most Percina larvae were collected in drift nets. Larval Percina species appeared in the drift before Etheostoma species in both study years. Application of molecular genetic and phenotypic tools to larval fish identification can advance understanding of larval darter ecology.