Aquatic hyphomycetes play a key role in organic matter processing in freshwaters. Traditionally, species have been identified through the morphology of their conidia, but mycelia can fail to sporulate, making aquatic hyphomycetes a group where DNA barcoding is crucial. We generated ITS barcodes for nine aquatic hyphomycetes without published ITS sequences that, together with all published sequences, were used to construct a neighbor-joining tree. In general, the new barcoded species grouped with species of the same genus, but Tricladium and Triscelophorus species were interspersed among different clades of the tree, confirming the multiple origins of aquatic hyphomycetes.