Allium crop germplasm is ephemeral vegetable due to its short-lived seeds. Application of the conventional low-temperature preservation is difficult to achieve long-term preservation. It is therefore of great significance to set up the cryopreservation procedure specific to the allium germplasm. In the present study, through the orthogonal experiment of three factors with four levels, we examined the cryopreservation methods using 30 accessions of four allium vegetables (Allium tuberosum Rottler ex Sprengle, Allium fistulosum L. var. giganteum Makino, Allium porrum L. and Allium cepa L.). These results was evaluated by the germination rate of the seeds after cryopreservation if compared to the germination rate of the seeds without treatments. The results showed significant differences among the ANOVA experiments (P<0.05), while the optimal procedure was the A1B4C1 (without cryoprotectant PVS2 treatment, storage in liquid nitrogen, and rewarming for 4 h at 40°C before testing the germination rate). We also determined the physiological indexes of superoxide dismutase (SOD), peroxidase (POD) activity, catalase (CAT) activity, malondialdehyde (MDA) content of cryopreservation seeds after seed rewarming. The MDA content of the seeds decreased after ultra-low temperature preservation of liquid nitrogen, while the SOD activity was higher than that of the control after the liquid nitrogen treatment. A decrease on the POD activity of A. tuberosum Rottler ex Sprengle, after liquid nitrogen treatment and an increase on the POD activity of A. porrum L., A. fistulosum L. var. giganteum Makino and A. cepa L. were detected. Collectively, these results provided optimal cryopreservation procedure for Allium vegetable germplasm resources.