Rice production in the Anhui province is threatened by fungal diseases. We obtained twenty-five fungal isolates from rice and wild rice leaves showing leaf spot disease collected along the Yangtze River. A phylogenetic analysis based on internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF1-α), and beta tubulin (TUB2) sequences revealed one isolate (SS-2-JB-1B) grouped with Nigrospora sphaerica, one (QY) with Nigrospora chinensis, twenty-two with Nigrospora oryzae, and one isolate (QY-2) grouped in its own clade, which are related to but clearly different from N. oryzae. Nineteen tested isolates, including sixteen strains from the N. oryzae clade and the three isolates of the other three clades, caused disease on detached rice leaves. The three isolates that did not belong to N. oryzae were also able to cause disease in rice seedlings, suggesting that they were rice pathogens. Isolate QY-2 differed from the other isolates in terms of colony morphology, cell size, and susceptibility to fungicides, indicating that this isolate represents a new species that we named Nigrospora anhuiensis. Our analysis showed that N. sphaerica, N. chinensis, and the new species, N. anhuiensis, can cause rice leaf spot disease in the field. This research provides new knowledge for understanding rice leaf spot disease.