The cereal grass barley was domesticated about 10,000 years ago in the Fertile Crescent and became a founder crop of Neolithic agriculture. Here, we report 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 revealed 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 in the past six millennia have not experienced a major lineage turnover although there is evidence for gene flow between cultivated and sympatric wild populations. We show the utility of ancient genomes from desiccated archaeobotanical remains in informing research into the origin, early domestication and subsequent migration of crop species.