In this study, we applied culture medium supplemented with 5.0, 15.0 and 30.0 mg/L As(Ⅴ) to grow four highland barley seedlings, ZQ5171, ZQ2000, ZQ16 and ZQ18 and characterized the As accumulation and distribution using hydroponics experiments and the treatment without As as control to investigate the arsenic effects on the plants. The results showed that after As(Ⅴ) treated the plant height, taproot length and biomass of ZQ5171 and ZQ2000 were inhibited, while the plant height, taproot length and biomass of ZQ16 and ZQ18 showed a phenomenon of “low promotion and high inhibition”. Compared with the control treatment, the plant height, taproot length and biomass of ZQ18 did not decrease significantly after As treated, and the root-shoot ratios were smaller and the ability of root-to-stem transfer of As was lower, suggesting ZQ18 was an As-tolerant variety. Meanwhile the growth of ZQ5171 and ZQ2000 were obviously poisoned, indicating ZQ5171 and ZQ2000 were As-sensitive varieties. The As contents in the roots, stems and leaves of highland barley decreased successively, which were 149.84-748.47, 1.81-234.71, 3.82-141.49 mg/kg, respectively. Except for ZQ18, As(Ⅴ) was the main form of As in the roots of the others highland barley, while As(Ⅲ) was the main form in the stems and leaves.