299 samples of highland barley were collected from Gansu, Tibet, Qinghai and Sichuan provinces, 143 mycotoxins were analysed by HPLC-MS/MS. The results showed that 13 mycotoxins in the samples from every province were not beyond the standard, just only T-2 Toxin and Zearalenone found. The detection rate of T-2 toxin in some areas of Qinghai was 49.26%; The detection rates of T-2 toxin, and zearalenone (ZEN) in some areas of Sichuan were 13.89% and 8.33% respectively; The detection rate of T-2 toxin in Gansu Province was 16.67%, which was not over the standard; The detection rate of ZEN in Tibet was 3.09%. According to the analysis data, the detection rates of T-2 toxin and ZEN were significantly different under different harvest methods and storage conditions (P < 0.05).