Three isolates of Phomopsis, causing shoot blight of peach, shoot tissue necrosis of plum, or bud death of Hosui Asian pear, respectively, were evaluated for their pathogenicity on apple, pear, peach, and plum. Current year's shoots of 1-year-old Stayman Winesap apple, Barlett pear, Babygold-7 peach, and Bruce plum trees were inoculated with each isolate by wounding a bud and applying agar blocks bearing young hyphae. The length of cankers on shoots was measured 10, 17, and 24 days after inoculation. Cankers developed on shoots of all hosts inoculated with the peach isolate and on peach shoots inoculated with plum and Asian pear isolates. No cankers developed on apple, pear, or plum shoots inoculated with plum and Asian pear isolates. In the first experiment, 10 days after inoculation, the length of cankers on apple trees (56.0 mm) inoculated with the peach isolate was not significantly different (P ≥ 0.05) from that on peach (42.8 mm), but was significantly greater than that on plum (25.3 mm) and pear (13.1 mm). The cankers on peach were significantly longer than those on pear, but not on plum. Cankers on all four hosts were significantly different from one another 17 and 24 days after inoculation. There was no significant difference between the length of cankers on peach shoots inoculated with plum and Asian pear isolates, and they were significantly smaller than those inoculated with the peach isolate. None of the control trees developed cankers. The three isolates differed in colony morphology, and appearance of conidiomata, conidiogenous cells, and α-conidia on potato-dextrose agar. None of the isolates produced β-conidia in culture. Multi-locus DNA fingerprint analysis and internal transcribed spacer sequence comparisons revealed similarities between the plum and Asian pear isolates but a significant difference between these two and the peach isolate. The results indicate that the Phomopsis sp. that causes shoot blight of peach has the potential to cause disease on other stone and pome fruits, and peach may also be susceptible to isolates of Phomopsis from different tree fruit hosts.