Vernalization, the requirement of a period of low temperature to induce transition from vegetative to reproductive state, is an important trait in grasses. The objective of this study was to identify the causative polymorphism(s) for the variation in vernalization requirement at the VRN1 locus in perennial ryegrass (Lolium perenne). Two allelic BAC clones of the VRN1 locus from the two genotypes Veyo and Falster with contrasting vernalization requirements were identified, sequenced, and characterized. The region surrounding the VRN1 locus in perennial ryegrass showed micro-colinearity to the corresponding region on chromosome 3 in Oryza sativa with conserved gene order and orientation, while the micro-colinearity to the corresponding region in Triticum monococcum was less conserved. Repeat elements constituted 42 and 58% of the Veyo and Falster BAC sequences, respectively, of which the majority showed similarity to the Athila-like Class I LTR retrotransposon Sabrina from Hordeum vulgare. Bioinformatic analysis of the allelic sequences identified a 8.6kb deletion in the first intron of the VRN1 gene in the Veyo genotype, which in a divergent selection experiment was associated with low vernalization requirement. Our findings suggest that the 8.6kb region in the first intron of the VRN1 gene is an important regulatory region for vernalization response in perennial ryegrass.