Multiple species of Colletotrichum can cause bitter rot disease of apple, but the identities and relative representation of the species causing the disease in Kentucky are unknown. A total of 475 Colletotrichum isolates were collected from diseased apples in 25 counties and characterized both morphologically and by using various molecular approaches. Four morphotypes corresponded to reported descriptions of bitter rot species. Morphotype 1, distinguished by the production of a pink color on potato dextrose agar (PDA), orange conidial masses, and fusiform spores, was consistent with C. acutatum. Morphotype 2, which produced gray or white mycelial colonies with orange conidial masses and fusiform spores, was also similar to C. acutatum. Morphotype 3 had abundant gray mycelium and rounded spores and was identical to C. gloeosporioides. Morphotype 4 produced ascospores and resembled Glomerella cingulata. Species-specific polymerase chain reaction (PCR) indicated that both Morphotype 1 and Morphotype 2 belonged to the C. acutatum species complex, whereas Morphotype 3 and Morphotype 4 corresponded to the C. gloeosporioides complex. Multigene sequence analyses revealed that sample isolates belonged to several newly erected species within these species complexes. Morphotype 1 was identified as C. fioriniae, which resides within the C. acutatum species complex. Morphotype 2 was identified as C. nymphaeae, which is also a species within the C. acutatum species complex. Some isolates of Morphotype 3 were identified as C. siamense and some as C. theobromicola; both species are grouped within the C. gloeosporioides species complex. Morphotype 4 was identified as C. fructicola, which is also placed within the C. gloeosporioides species complex. C. fioriniae was the most common species causing bitter rot in Kentucky, comprising more than 70% of the isolates. Molecular fingerprinting using random amplified polymorphic DNA (RAPD) suggested that isolates within C. fioriniae belonged to a relatively homogeneous population, while isolates within C. siamense, C. theobromicola and C. fructicola were more diverse. Infectivity tests on detached fruit showed that C. gloeosporioides species-complex isolates were more aggressive than isolates in the C. acutatum species complex. However, isolates within the C. acutatum species complex produced more spores on lesions compared to isolates within the C. gloeosporioides species complex. Aggressiveness varied among individual species within a species complex. C. siamense was the most aggressive species identified in this study. Within the C. acutatum species complex, C. fioriniae was more aggressive than C. nymphaeae, causing larger, deeper lesions. Apple cultivar did not have significant effect on lesion development. However, Colletotrichum species produced more spores on Red Stayman Winesap than on Golden Delicious. Fungicide sensitivity tests revealed that the C. acutatum species complex was more tolerant to thiophanate-methyl, myclobutanil, trifloxystrobin, and captan compared to the C. gloeosporioides species complex. The study also revealed that mycelial growth of C. siamense was more sensitive to tested fungicides compared to C. fructicola and C. theobromicola. These research findings emphasize the importance of accurate identification of Colletotrichum species within each species complex, since they exhibit differences in pathogenicity and fungicide sensitivity.