ABSTRACT.- Paspalum L. is a genus of the Poaceae family, with many species serving as well-adapted forage plants in subtropical climates and continuous grazing systems. However, Claviceps paspali, an ascomycete of the order Hypocreales, represents a major threat to Paspalum species. This fungus induces ergot disease, characterized by the replacement of infected flower seeds with sclerotia, which adversely affects seed production and animal health through mycotoxin production. Although ergot disease is reported in many countries, no totally resistant Paspalum cultivars have been reported for commercial use. This study comparatively evaluated disease development in six Paspalum species under greenhouse conditions with three specific isolates of C. paspali, along with field trials. In addition, field isolates of C. paspali were analyzed for phenotypic characteristics and phylogenetic relationships. Greenhouse evaluation revealed variable susceptibility among Paspalum species, with P. malacophyllum,P. notatum and P. umbrosum being the most resistant. In field trials P. dilatatum showed the highest severity index, while P. umbrosum and P. malacophyllum were less affected. Phenotypic characterization of 22 C. paspali isolates showed variability in pigmentation and growth rates, with PDA being the culture medium where the highest growth rate was observed. Phylogenetic analysis of the isolates identified two well-supported lineages between C. paspali species. This research reports an ergot resistance gradient among Paspalum species, identifying genuine sources of resistance. In addition, virulent isolates of C. paspali potentially useful for rapid screening of accessions or crosses in Paspalum breeding programs were identified. © 2024 The American Phytopathological Society