Abstract: A new procedure, so called two-step protection for cryopreservation of Azolla meristems was developed, i. e. the meristems were pretreated by using a low concentration of DMSO, which exhibit strong penetrating ability before freezing, then treated by a high concentration of VS2, which has lower penetrating ability, as a protectant while freezing. The effect of the procedure on the meristems both of A. filiculoides and A. caroliniana at the same cooling rate was investigated. All physiological state of the meristems, concentration of DMSO, time course of protection treatment and cooling rate can effect significantly on survival rate of the meristems. 53 meristems of A. filiculoides from 18 treatments frozen at-30℃ could survive,the best survival rate reached 100%, and 14 meristems of A. filiculoides from 5 treatments frozen at-32.5 ℃ could survive as well.