Timothy (Phleum pratense L.) is the major forage species within the genus Phleum and is widely grown in cool-season regions of the world, including the Nordic countries. Most temperate perennial grasses have a dual induction requirement for flowering. Primary induction is achieved by a prolonged period of low temperature, i.e., vernalization, and/or short days, while secondary induction requires transition to long days and increased temperatures. While induction of flowering in timothy requires secondary induction, a vernalization response has not previously been reported for this species. We studied the vernalization response in 38 genotypes of diverse geographic origin and found that vernalization accelerated heading in all genotypes. In addition, considerable variation in the vernalization response was observed between genotypes and a requirement for vernalization to induce flowering was indicated in genotypes of Northern origin. While heading time in general was found to be correlated to geographic origin of genotypes, a strong vernalization response was identified in genotypes of contrasting geographic origin. Candidate genes for the vernalization response genes VRN1, VRN2 and VRN3 (also named FT) were identified and transcript analysis during primary and secondary induction showed that VRN1 transcription was induced by vernalization. However, when not vernalized, genotypes with a strong vernalization response showed a prolonged repression of VRN1 transcription throughout secondary induction. In contrast, in genotypes with a mild vernalization response VRN1 transcription was induced by transfer to secondary induction. We conclude that significant genetic variation for the vernalization response is present within timothy and suggest that differential regulation of VRN1 transcription discriminates genotypes with contrasting vernalization responses.