Alfalfa (Medicago sativa L.) is one of the most popular forage legume crops worldwide. Its cultivation in the boreal and sub-boreal zone is restricted by inadequate winter hardiness, but global warming may increase its adaptability in these latitudes. Here, we examined variation in growth and freezing tolerance of four alfalfa cultivars recommended for the northern temperate climates of Europe (Alexis, Lavo, Live, and Nexus) and two cultivars with adaptation to milder or Mediterranean climates (Rangelander and Hunter River). Two experiments under controlled conditions (growth cessation and cold acclimation experiments) along with a 2-yr field experiment were conducted. Lavo was the most freezing-tolerant cultivar in both the cold acclimation and field experiments. Both Rangelander and Hunter River showed poor freezing tolerance. Lavo responded to decreasing temperatures, unlike the response to shorter day length, by allocating biomass to the roots. In general, better freezing tolerance was associated with high total nonstructural carbohydrate and low starch content. The field experiment results revealed that the more freezing-tolerant cultivars may have some advantages regarding yield, especially in the second year, but the differences between the cultivars were modest.