Seventy-two Colletotrichum isolates from peach, apple, pecan, and other hosts were examined morphologically and tested in vitro for benomyl sensitivity and for polymorphisms in the ribosomal 188 and 28S transcriptional unit. In general, the isolates separated into pink and gray colony types. Each type was isolated from peach, apple, and pecan. Regardless of host origin, pink isolates produced pink to pinkish-orange colonies on potato-dextrose agar (PDA), grew on benomyl-amended medium, produced mostly fusiform conidia, and had similar restriction fragment length polymorphism (RFLP) patterns. Gray isolates produced gray colonies on PDA, grew slightly or not at all on benomyl-amended medium, had conidia with rounded ends, and had similar RFLP patterns that were distinct from pink isolates. Symptoms on detached peach fruit following inoculation with gray and pink isolates were not visually distinguishable. The two types corresponded to C. acutatum (pink isolates) and C. gloeosporioides (gray isolates), respectively. We concluded that C. acutatum and C. gloeosporioides are separate species and are found on peach, apple, and pecan.