Biodiversity hotspots have provided useful geographic proxies for conservation efforts. Delineated from a few groups of animals and plants, biodiversity hotspots do not reflect the conservation status of freshwater fishes. With hundreds of new species described on a yearly basis, fishes constitute the most poorly known group of vertebrates. This situation urges for an acceleration of the fish species inventory through fast and reliable molecular tools such as DNA barcoding. The present study focuses on the freshwater fishes diversity in the Sundaland biodiversity hotspot in Southeast Asia. Recent studies evidenced large taxonomic gaps as well as unexpectedly high levels of cryptic diversity, particularly so in the islands of Java and Bali. The Cypriniformes genera Rasbora and Nemacheilus account for most of the endemic species in Java and Bali, however their taxonomy is plagued by confusion about species identity and distribution. This study examines the taxonomic status of the Rasbora and Nemacheilus species in Java, Bali and Lombok islands through DNA barcodes, with the objective to resolve taxonomic confusion and identify trends in genetic diversity that can be further used for conservation matters. Several species delimitation methods based on DNA sequences were used and confirmed the status of most species, however several cases of taxonomic confusion and two new taxa are detected. Mitochondrial sequences argue that most species range distributions currently reported in the literature are inflated due to erroneous population assignments to the species level, and further highlight the sensitive conservation status of most Rasbora and Nemacheilus species on the islands of Java, Bali and Lombok.