A study of genetic variation in freezing tolerance of Eucalyptus globulus was conducted in winter by subjecting leaf discs from nursery-grown seedlings to artificial freezing at temperatures ranging from –5.5 to –10.0°C. A total of ~6200 seedlings from 477 open-pollinated families from the full natural range were assessed in three separate experiments. Patterns of frost tolerance with race were not clear and consistent in all three experiments, although inland south-eastern Tasmanian and West Coast Tasmanian races were usually the more frost tolerant (T₅₀ = –8.8°C, cf. overall mean of –8.3°C). Poor correlations between experiments also made clear identification of superior localities difficult. Winter-frost tolerance was a trait with considerable variation and also under strong additive genetic control. Estimated heritabilities of the relative electrical conductivity following a set freezing temperature and the calculated trait T₅₀ were in the range of 0.27–0.71, with small standard errors. Predicted breeding values in all experiments indicated that the best families were tolerant of ~1.4°C colder temperatures than average. Seedlings appeared equally capable of rapid dehardening, when average T₅₀ cold hardiness changed from –10.5 to –5.7°C during a 10-day period, or ~3–4°C per week. The issue of more thoroughly assessing the genetic basis of frost tolerance in E. globulus through field-testing is discussed.