The preparation of highland barley grain samples using conventional transmission electron microscope (TEM) methods often results in sample preparation failure, making it impossible to observe the grain ultrastructure. This is due to the dense structure, thick cell walls, and poor permeability of highland barley seeds. To address these issues, high pressure treatment was introduced during the glutaraldehyde fixation and resin penetration steps. Additionally, the concentrations of the glutaraldehyde fixative solution, osmium acid fixative solution, and resin soaking solution were adjusted to suit the characteristics of highland barley grains. The samples were observed under different reagent concentrations and high pressure treatment conditions. By comparing the differences, the optimal reagent concentrations and high pressure ranges were selected, and the suitable experimental conditions were further refined through orthogonal experiments.The optimal pre-treatment conditions for TEM of highland barley grains were determined as follows:glutaraldehyde volume fraction of 4.5%, osmium acid concentration of 15g/L, acetone to epoxy resin volume ratio of 1∶1 in the resin soaking solution treated overnight, high pressure treatment of 50MPa for both glutaraldehyde fixation and resin soaking, and a treatment time of 10 minutes. Under these conditions, the epidermal cell wall of highland barley grains remained intact without breakage. The aleurone layer structure was complete and clear, with no cavities. The cell structure of the endosperm layer was complete and regularly shaped, and the starch grains were intact and unwrinkled. These conditions are optimal for preparing high-quality transmission electron microscopy samples of highland barley grains.