Anthracnose is a destructive disease that affects a wide range of crop plants especially in tropical and subtropical regions. Colletotrichum spp. are the major pathogens causing anthracnose. In this study, we collected and identified the pathogen from diseased samples of Stylosanthes, a major tropical forage crop. The ability of the pathogen to naturally infect Arabidopsis thaliana was examined. Sequence analysis of ITS, ACT, CHS, and GAPDH genes showed the pathogen to be Colletotrichum gloeosporioides sensu lato (s.l.), and this was supported further by morphological characterization of representative isolates. The disease symptoms and cellular infection process of aggressive isolates (DZ‐19 and HK‐04) and a weak isolate (CJ‐04) were compared. DZ‐19 and HK‐04 caused more severe disease symptoms on both young seedlings and adult plants of Col‐0 and Ws‐2 ecotypes compared to CJ‐04. Furthermore, the more aggressive isolates showed faster and earlier germination of conidia, formation of appressoria, and growth and development of hyphae during the infection. Genetic analysis of the defence response and expression profiling of defence marker genes demonstrated the involvement of MAP kinase, Ca2+‐dependent protein kinase, salicylic acid, ethylene, and jasmonic acid pathways in the resistance against anthracnose. These results suggest that the Arabidopsis–Colletotrichum gloeosporioides pathosystem should provide a valuable tool for exploring the resistance mechanisms against this pathogen. A pathosystem between Arabidopsis and C. gloeosporioides sensu lato from Stylosanthes has been established, which could be an effective tool for studying the resistance mechanisms of anthracnose.