Vernalization promotes flowering in winter wheat cultivars, whereas spring wheat cultivars are able to transition from vegetative to reproductive phase without vernalization. The wheat vernalization requirement is mainly controlled by the major locus Vrn-1, an APETALA1/FRUITFULL MADS-box gene homolog. To study natural variation of the vernalization requirement in a wild progenitor of common wheat, we sequenced the Vrn-D(t)1 locus in four accessions of Aegilops tauschii Coss. Some structural mutations were found in the promoter and first intron regions of Vrn-D(t)1, and haplotype analysis was conducted to examine the distribution of each identified mutation within 211 accessions of Ae. tauschii germplasm. Out of the total, nine accessions, which were originally collected in Afghanistan and Pakistan, contained deletions of a 5.4-kb sequence in the critical region of the Vrn-D(t)1 first intron. The 5.4-kb deletion mutation appeared independently of the dominant allele of the common wheat Vrn-D1 locus. The large deletion was absolutely associated with a lack of vernalization requirement for flowering under long-day conditions, but had no influence on heading date under field growth conditions. The levels of Vrn-1 and WFT transcript increased in the Ae. tauschii accessions having the large deletion. Identification of natural mutant accessions with a loss of vernalization requirement indicates the agricultural significance of Ae. tauschii as a genetic resource for wheat breeding.