Members of Plectosphaerella inhabit different substrates, including plants, soil and insects, and most species are pathogens causing large losses in agriculture. During a survey of endophytic fungi in aquatic plants in southwest China, 112 strains of Plectosphaerella were isolated, representing two new species, P. endophytica sp. nov. and P. sichuanensis sp. nov., as well as two known species, P. cucumerina and P. pauciseptata . The novel taxa are described and illustrated here using combined morphological and multi-locus phylogenetic (LSU-ITS-TEF-1α-TUB2) analyses. Our result revealed Plectosphaerella species inhabiting within aquatic plants in southwest China, and the separation frequency of each species was presented.