Technical Abstract: Anthracnose of soybean is caused by several Colletotrichum species. Petiole samples were collected from Alabama, Illinois, and Mississippi. Diseased tissues suspected of being caused by Colletotrichum species were cut into 1-2 cm in lengths, surface-disinfested, and placed on water agar. Pure cultures obtained by picking single spores from sporulating acevuli on tissue or hyphal tips on agar were transferred to acidic potato-dextrose agar (APDA) at 24 ± 1°C under 12 h cool-white fluorescent lighting. Isolates were grouped by their morphological characteristics. One group consisting of six isolates (4 from IL and 1 each from AL and MS) was identified as C. chlorophyti. Soybean plants (cv.Williams 82) at the V2-V3 stage were atomized with a suspension of fragmented mycelia (40 mg/mL) using one isolate from IL. Plants were kept in a moisture chamber with > 90% relative humidity for 48 h in the dark, and then transferred to normal growing conditions. Three days post-inoculation, necrosis of apical tissues and at the edge of young leaves and petioles were observed. Further comparisons based on sequence analyses of the partial ITS rDNA region, actin, ß-tubulin, GAPDH, and histone 3 genes resulted in = 96% identity of all the 5 sequences to two C. chlorophyti isolates. This is the first report of C. chlorophyti in the United States, and of it infecting soybean.