Highland barley was fermented with Cordyceps militaris , Stropharia rugoso-annulata , Morchella esculenta , Schizophyllum commune and Tremella sanguinea . The flavor profiles were investigated by electronic nose (E-nose), headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) and sensory evaluation by train panel. Fermentation with mushroom mycelium was able to change the aroma profile of highland barley. The original strong grassy taste was reduced due to a decrease in hexanal, decanal and 2-pentylfuran, and new aromatic flavors (floral, sweet and mushroom fragrance) were acquired after fermentation. The overall flavor of the fermented highland barley varied with mushroom strains. Schizophyllum commune gave a heavier sour taste to the fermented highland barley. However, fermentation with T. sanguinea increased the content of methyl 4-methoxybenzoate making the sample difficult to accepted. Fermentation with C. militaris , M. esculenta , and S. rugoso-annulata increased the volatile contents. The high levels of 1-octen-3-ol and esters gave a strong mushroom, oily and fruity flavor. Morchella esculenta showed the best performance and the highest acceptance in the fermented highland barley. Our results suggest that fermentation with mushroom mycelium can improve the flavor of highland barley, which provides an innovative utilization of highland barley.