Breeding alfalfa (Medicago sativa L.) with superior freezing tolerance could be accelerated by the identification of molecular markers associated to that trait. Dehydrins are a group of highly hydrophilic proteins that have been related to low temperature tolerance. We previously identified a dehydrin restriction fragment length polymorphism (RFLP) among populations recurrently selected for superior tolerance to freezing (TF). Analysis of crosses between genotypes with (D+) or without (D-) that RFLP revealed a significant impact on freezing tolerance. In this study, we sought to develop a PCR marker for freezing tolerance based on prior evidence of a relationship between size variation in Y(2)K(4) dehydrins and the RFLP. Results confirm the enrichment of Y(2)K(4) sequences of intermediate size (G2 group) in response to recurrent selection and in the D+ progeny. Analysis of genomic sequences revealed significant intron-length polymorphism (ILP) within the G2 group. G2 sequences with a characteristic short intron were more frequently found in D+ genotypes. Amplification using sequence-characterized amplified region (SCAR) primers bordering the intron confirmed an increase in the number of fragments with small introns in the D+ progeny and in the ATF5 population obtained after five cycles of recurrent selection for superior TF within the cultivar Apica (ATF0). Conversely, there was a reduction in the number of fragments with long introns in the D+ progeny and in ATF5 as compared to ATF0. Recurrent selection for superior tolerance to freezing in combination with ILP identified a sequence variant of Y(2)K(4) dehydrins associated to the phenotypic response to selection.