Natural variation for wheat vernalization requirement is mainly controlled by the Vrn1, Vrn2, Vrn3 and Vrn4 genes. The first three genes have already been cloned, and allelic variants were found at the different homeologous, associated with differences in flowering time. Less is known about Vrn4, which has been detected only in the centromeric region of chromosome 5D (Vrn-D4), and linked to the Vil1 gene. Arabidopsis Vil genes play an important role in vernalization, and wheat Vil1 transcript levels were shown to be significantly up-regulated by vernalization. We constructed a high-density genetic map of the Vrn-D4 region (3,182 gametes), including 22 molecular markers, and mapped Vrn-D4 within a 0.09 cM interval in the centromeric region of chromosome 5D. This region was found to be collinear with a 2.24 Mb region of Brachypodium chromosome 4, including 303 predicted genes, 11 of which have been annotated as related to developmental processes. Two recombination events separated wheat Vil-D1 from Vrn-D4 confirming that this gene is not a candidate for Vrn-D4. We are currently analyzing the 11 linked candidate genes for polymorphisms between the parental lines of our mapping population. We used the Vrn-D4 molecular markers identified in this study to test the epistatic interactions between Vrn-D4 and other vernalization genes. We detected highly significant (P<.0001) epistatic interactions between Vrn-D4 and Vrn-A1, Vrn-B1, Vrn-D1, and Vrn-B3 confirming that Vrn-D4 is part of the vernalization pathway and that it likely operates upstream of the regulatory feedback loop involving Vrn-1, Vrn-2 and Vrn3.