Archaeobotanical materials subject to aDNA analysis were recovered from Yumuktepe and Yenikapi, two important archaeological sites in Anatolia and date back to the Pottery Neolithic Period i.e., 7th millennium BC. Many charred ancient seeds representing various cereal species including a great number of wheat grains were documented in mentioned sites. Among the cereal seeds, charred wheat samples were tentatively identified as Triticum aestivum subsp. spelta L. or Triticum new glume wheat (NGW) or atypical emmer or naked wheat in Yumuktepe and Yenikapi showed similarities with the morphological characteristics of T. aestivum subsp. spelta wheat, but it was difficult to reach a firm conclusion. This study aimed to provide genetic data to enable more precise identification of charred wheat seeds using an ancient DNA (aDNA) approach. aDNAs were successfully extracted from the representative charred seeds of T aestivum subsp. spelta or NGW or atypical emmer or naked wheat. The PCR amplification of 26SrDNA and IGS gene regions with aDNA was carried out and sequenced. The expected product sizes of IGS 158 bp for the D genome and 87 bp for the A or B genomes and DNA sequence comparisons with other wheat species revealed that T. aestivum subsp. spelta or NGW or atypical emmer or naked wheat samples included the D genome from Aegilops tauschii and is more likely to be T. aestivum subsp. spelta. The discovery of T. aestivum subsp. spelta grains in the Yenikapi and Yumuktepe suggest that the cultivation of hexaploid wheat was widespread. Further, spelta hulled wheat, which is the progenitor of the hexaploid wheat, might have been cultivated in these settlements.