We report a successful extraction and sequencing of ancient DNA from carbonized rice grains (Oryza sativa) from six archaeological sites, including two from India and four from Thailand, ranging in age from ca. 2500 to 1500 BP. In total, 221 archaeological grains were processed by PCR amplification and primary-targeted fragments were sequenced for comparison with modern sequences generated from 112 modern rice populations, including crop and wild varieties. Our results include the genetic sequences from both the chloroplast and the nuclear genomes, based on four markers from the chloroplast and six from the nuclear genome. These markers allow differentiation of indica rice from japonica rice, the two major subspecies of Asian rice (O. sativa) considered to have separate geographical origins. One nuclear marker differentiates tropical and temperate forms of subspecies japonica. Other markers …