Bitter rot, caused by Colletotrichum spp., is one of the most important diseases in apple-growing areas of the world, and is the most difficult summer apple disease to manage in Illinois. Studies have shown that there is considerable genetic and molecular variability within isolates of Colletotrichum spp. The accurate identification of Colletotrichum spp. within the species complex is therefore critical since species exhibit differences in pathogenicity and sensitivity to fungicides. A species-specific diagnosis is very important for developing an effective disease management strategy. Orchard surveys were conducted in Illinois in the 2019 and 2020 seasons and infected apples were collected from 10 different cultivars in 28 orchards. The pathogens were isolated using potato dextrose agar (PDA). After performing DNA extraction from conidia, PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using the primer GDF1 was done. Sequencing and subsequent blast in NCBI GenBank positively identified the isolates as C. fioriniae, which reside within the C. acutatum species complex, and C. siamense, belonging to the C. gloeosporioides species complex. Preliminary analysis showed that C. fioriniae is the most common species causing bitter rot in Illinois apple orchards, comprising more than 60% of the isolates. Both species were found in the same orchard, and in some cases, on the same tree.