Molecular data from 35 of the 50 Acrossocheilinae species suggest that the species-level diversity in the subfamily has been overestimated, likely due to inadequate taxon and geographic sampling and reliance on morphological characters that vary intraspecifically. Three new genera, one resurrected genus, two resurrected species, and one new species are diagnosed and described herein. Nine synonyms of three valid species of Acrossocheilinae are recognized from the Yangtze, Xijiang, Song Hong, Annamite, and Mekong ecoregions in East and Southeast Asia. Thirty-two valid and six putative new species are indicated by molecular data and a key to the genera is provided. As more molecular and morphological data become available, additional taxonomic changes in this widespread and generally poorly known subfamily are likely.