The cereal grass barley was domesticated about 10,000 years before the present in the Fertile Crescent and became a founder crop of Neolithic agriculture. Here we report the genome sequences of five 6,000-year-old barley grains excavated at a cave in the Judean Desert close to the Dead Sea. Comparison to whole-exome sequence data from a diversity panel of present-day barley accessions showed the close affinity of ancient samples to extant landraces from the Southern Levant and Egypt, consistent with a proposed origin of domesticated barley in the Upper Jordan Valley. Our findings suggest that barley landraces grown in present-day Israel have not experienced major lineage turnover over the past six millennia, although there is evidence for gene flow between cultivated and sympatric wild populations. We demonstrate the usefulness of ancient genomes from desiccated archaeobotanical remains in informing research into the origin, early domestication and subsequent migration of crop species.