Scenarios of climate changes indicate longer and more frequent spells of mild weather during winter in northern latitudes. De-hardening in perennial grasses could increase the risk of frost kill. In this study, the resistance to de-hardening of different grass species and cultivars was examined, and whether the resistance changes during winter or between years, was tested. In Experiment 1, two cultivars of timothy (Phleum pratense L.) and perennial ryegrass (Lolium perenne L.) of contrasting winter hardiness were grown under ambient winter conditions, transferred from the field in January and April 2006 to the laboratory for 9 d with controlled de-hardening conditions of 3°C, 9°C and 15°C. The timothy cultivars were tested at 3°C, 6°C and 9°C in a similar experiment (Experiment 2) in January 2007. De-hardening, measured as decrease in frost tolerance (LT50), was less in timothy than in perennial ryegrass and increased with increasing temperatures. The northern winter-hardy cultivar Engmo of timothy de-hardened more rapidly than the less-hardy cultivar Grindstad, but had higher initial frost tolerance in both experiments, whereas there was less difference between cultivars of perennial ryegrass in Experiment 1. Cultivar Grindstad of timothy lost all hardiness in early spring at all temperatures, whereas cultivar Engmo maintained some hardiness at 3°C. Cultivar Engmo de-hardened at a lower rate in 2007 than in 2006, in spite of similar frost tolerance at the start of de-hardening treatment in both years. This indicates that the rate of de-hardening was controlled by factors additional to the initial frost tolerance and that autumn weather conditions might be important for the resistance to de-hardening.