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Helicosporous hyphomycetes are a morphologically fascinating group of Tubeufiales. Ten dematiaceous helicosporous asexual morphs and two sexual morphs collected from aquatic habitats are characterized in this study using morphological features and phylogenetic analyses. Four new species of Neoacanthostigma, viz. N. aquaticum, N. brunneisporum, N. guangxiense and N. latisporum, are described and illustrated. The phylogenetic analyses of combined ITS, LSU and TEF1α sequence data place all taxa in the genus Neoacanthostigma (Tubeufiaceae, Tubeufiales) and provide evidence to support the establishment of the new taxa. The differentiating morphological characters of Neoacanthostigma with other helicosporous species are compared and discussed. A key to all Neoacanthostigma species is also provided.