Neohelicomyces species are a group of helicosporous hyphomycetes with the potential to produce secondary metabolites. During our investigation of helicosporous fungi, six collections were isolated from both terrestrial and freshwater habitats in Guizhou Province, China. Based on multigene phylogenetic analysis (ITS, LSU, tef1α and rpb2), coupled with morphological data, three new Neohelicomyces species, viz. N. guizhouensis, N. helicosporus and N. hydei were established. A list of accepted Neohelicomyces species with molecular data was provided. The strain of Neohelicomyces pallidus (UAMH 10535) was synonymised under N. denticulatus based on molecular data.