AbstractPseudostellaria heterophylla, a valuable Chinese herbal medicine, is widely cultivated in China, with a planted area of 5402.7 hm2 in Shibing County, Guizhou Province. The occurrence of leaf spot disease represents a significant challenge in the production area. In this study, 30 samples of P. heterophylla exhibiting symptoms of leaf spot were collected from three primary production areas in Guizhou Province. Following isolation and purification, 51 isolates were classified into two genera based on both cultural and conidial characteristics, along with ITS sequence data. Fourteen representative strains were selected for morphological and multigene molecular identification, which revealed that these representative strains were affiliated with Alternaria arborescens species complex (SC), Alternaria tenuissima, Alternaria gaisen, and Sclerotiophoma versabilis. The isolation rates of Alternaria and Sclerotiophoma were 84.3% and 15.7%, respectively. Pathogenicity tests confirmed the identity of the causal agents and demonstrated varying levels of aggressiveness among the different genera and species of pathogens towards P. heterophylla leaves. This study marks the first identification of A. arborescens SC, A. gaisen, and S. versabilis as pathogens causing leaf spot in P. heterophylla in China.