To advance barley research, we produced a TILLING (Targeting Induced Local Lesions IN Genomes) population of the sixrowed barley cultivar ‘Xiyin2’. Through ethyl methane sulfonate(EMS) mutagenesis treatment, we established a 3 750 mutagenised M₁ population and a 2 012 mutagenised M₂ population. Totally phenotypic variations in 17.35% of the M₂ population were observed. In which 115 lines showed chlorosis, 98 lines showed dwarfing, 101 lines showed very early/late flowering, and others 35 lines showed less tilling, small leaves, or produced no seeds. Likewise, genomic DNA was extracted from the M₂ lines for studying the mutation frequency. The estimated mutation frequency was one mutation per 661 kb by CELⅠbased screening, it is the same range as the previously studies. During our investigation, 3 different mutations in the EDR1 (AF305912) gene were identified, among which the Pro to Ser transition happened in extron region. The ‘Xiyin2’ TILLING population will be an important resource in the development of barley with specific characters.