As one of the world’s earliest domesticated crops, barley is a model species for the study of evolution and domestication. Domestication is an evolutionary process whereby a population adapts, through selection; to new environments created by human cultivation. We describe the genome-scanning of molecular diversity to assess the evolution of barley in the Tibetan Plateau. We used 667 Diversity Arrays Technology (DArT) markers to genotype 185 barley landraces and wild barley accessions from the Tibetan Plateau. Genetic diversity in wild barley was greater than in landraces at both genome and chromosome levels, except for chromosome 3H. Landraces and wild barley accessions were clearly differentiated genetically, but a limited degree of introgression was still evident. Significant differences in diversity between barley subspecies at the chromosome level were observed for genes known to be related to physiological and phenotypical traits, disease resistance, abiotic stress tolerance, malting quality and agronomic traits. Selection on the genome of six-rowed naked barley has shown clear multiple targets related to both its specific end-use and the extreme environment in Tibet. Our data provide a platform to identify the genes and genetic mechanisms that underlie phenotypic changes, and provide lists of candidate domestication genes for modified breeding strategies.