The causal agent of tobacco anthracnose was identified as Colletotrichum destructivum based on the morphology of the fungus and a comparison of the sequence of the rDNA ITS with those of other Colletotrichum species. The infection process on tobacco (Nicotiana tabacum and N. benthamiana) was examined by light microscopy, which revealed that the pathogen acted as an intracellular hemibiotroph. Penetration occurred preferentially at the anticlinal walls of epidermal cells by an appressorium and penetration peg. An infection vesicle formed in the penetrated host cell by 48 h after inoculation, and out of this, a multi-lobed infection vesicle grew which remained limited to the initially infected cell. The interaction at this point was biotrophic, which was confirmed by plasmolysis and accumulation of a vital stain by the infected host cells. Thin secondary hyphae arose from multi-lobed infection vesicles at 60 h after inoculation, which then penetrated the host cell wall and began the necrotrophic phase of the infection. Acervuli formed on the plant surface by 96 h after inoculation, typically with a single melanized seta. In addition to tobacco, the fungus could infect alfalfa, cowpea, and Medicago truncatula, but not soybean. The process of infection of C. destructivum in tobacco was very similar to that previously reported in alfalfa and cowpea.