To screen high density and evenly distributed molecular markers on individual barley chromosomes, we used a total of 2 267 primer pairs previously developed and amplified in CS, barley (Golden promise) and wheatbarley disomic addition lines (2H, 3H, 4H, 5H, 6H and 7H). We assigned 534 IT markers to seven barley chromosomes (1H7H) using 6 wheatbarley disomic addition lines: 96 to 1H, 84 to 2H, 60 to 3H, 105 to 4H, 59 to 5H, 80 to 6H, and 50 to 7H, respectively. Further using the Triticeae multiomics center and the barley reference sequence, except for molecular markers CINAU800, CINAU1734, CINAU1796, CINAU1736 and CINAU1691, it was found that gene sequences corresponding to all of other molecular markers could align to the barley reference sequence. This result indicated that 534 specific markers for individual chromosomes of barley were screened and the polymorphic rate (23.56%) was slightly higher than that of other molecular markers. This result further confirmed that these specific markers for individual chromosomes of barley could be used to trace the individual chromosome of barley.