In northern temperate zones, peach trees are vulnerable to cold temperature injury in the fall, particularly as climate change prolongs warm weather in the fall and potentially delays the onset of cold acclimation. Four experiments evaluated how cold acclimation of flower buds and shoot phloem, cambium, and xylem is affected by exposure to varying temperatures in the fall. One-year-old peach shoots from trees grown in Maine, USA, were collected from October through November, exposed to 1, 3, or 6 days of low, high, and freezing temperatures, and subjected to stepwise controlled freezing to about −30 °C. Injury was visually quantified as oxidative browning of flower buds and shoot tissues. High temperature exposure, even of a single day, decreased cold tolerance of flower buds and shoot tissues until late November, when high temperatures only minimally decreased cold hardiness. In mid-November, increasing the duration of high temperature exposure from 1 to 3 days decreased cambium and phloem hardiness, but hardiness in flower buds was not further decreased by the longer duration of 3 days. By late November, hardiness in flower buds, cambium, and phloem was less responsive to high temperature, and was increased by prior exposure to 6 days of freezing. After high temperature, xylem lost hardiness to a small degree in mid-October and late November, but in mid-November this occurred in only one experiment. In this study, deacclimation during high temperature in the fall was greater in cambium and phloem than in flower buds and at times greater than in xylem.