Aims To characterize causal pathogen of Sansevieria trifasciata anthracnose through morphology and molecular analysis; to evaluate the host range of the pathogen; and to explicate the infection process by the pathogen histopathologically. Methods and Results Symptomatic leaves of S. trifasciata were collected from five states in Malaysia. The causal pathogen was isolated and identified for the first time in Malaysia as C. sansevieriae based on morphological and multi-gene phylogenetic analyses using ITS, TUB2 and GAPDH sequences. Pathogenicity tests were conducted on different hosts. Colletotrichum sansevieriae was not pathogenic towards S. cylindrica, S. masoniana, Furcraea foetida, Chlorophytum comosum, Aloe vera and Gasteria carinata, confirming the exceptionally high host specificity for a species of Colletotrichum. Histopathology was performed using light microscope and scanning electron microscopy to study the infection process of C. sansevieriae on S. trifasciata. Colonization of host leaves by the pathogen was observed 2 days after inoculation. Conclusions Colletotrichum sansevieriae caused anthracnose of S. trifasciata in Malaysia. It is a host-specific pathogen and colonized the host intracellularly. Significance and Impact of the Study This is the first report of C. sansevieriae causing anthracnose of S. trifasciata in Malaysia. The host range test and understanding of the infection process will provide better understanding of the host-pathogen relationship and beneficial for effective disease management.