In diploid wheat (Triticum monococcum), and likely in other Triticeae species, the VRN1 gene is essential for the initiation of the reproductive phase, and therefore, a detailed characterization of its regulatory regions is required to understand this process. A CArG-box (MADS-box-binding site) identified in the VRN1 promoter upstream from the transcription initiation site has been proposed as a critical regulatory element for the vernalization response. This hypothesis was supported by the genetic linkage between CArG-box natural deletions and dominant Vrn1 alleles for spring growth habit and by physical interactions with VRT2, a MADS-box protein proposed as a putative flowering repressor regulated by vernalization. Here, we describe a T. monococcum accession with a strong vernalization requirement and a 48-bp deletion encompassing the CArG-box in the VRN1 promoter. Genetic analyses of 2 segregating populations confirmed that this VRN1 allele is completely linked with a strong winter growth habit (vrn-A(m)1b). Transcript levels of the VRN1 allele with the 48-bp deletion were very low in unvernalized plants and increased during vernalization to levels similar to those detected in other wild-type vrn-A(m)1 alleles. Taken together, these results indicate that the CArG-box found upstream of the VRN1 transcription initiation site is not essential for the vernalization response.