To avoid publishing twice, the original abstract will be published with the other “Southern Division 2019” abstracts in Phytopathology. Colletotrichum fungi cause apple, strawberry and blueberry fruit rots, which result in significant losses for Kentucky growers. As mixed-fruit orchards become common, the potential for cross-infection has management implications. Our first objective was to characterize Colletotrichum strains from different fruits in Kentucky orchards to determine host specificity. Multigene sequence analysis revealed that C. nymphaeae was most frequently found on strawberry, while C. fioriniae was the only species on blueberry. Both were previously reported on apple. We performed fruit inoculations with a representative group of isolates. Regardless of original host, all isolates caused disease on all fruits. Accurate species identification is critical for management because Colletotrichum spp. differ in fungicide sensitivity, but sequencing methods are too slow for routine diagnosis. Our second objective was to evaluate sequences commonly used for multigene identification to determine if a single gene could accurately identify the species. Multiple isolates were identified by standard multigene sequencing, and whole genomes of a representative group of isolates were compared to individual gene trees. This analysis revealed that two genes, CAL and CHS, are necessary for species and subgroup identification. These findings will help us to develop more effective diagnostic and management procedures for Colletotrichum diseases in Kentucky mixed-fruit orchards.