The embryogenic callus of nematode-resistant Pinus massoniana was used to explore the effects of various factors of cryopreservation on the ultra-low temperature preservation. The three factors were determined by orthogonal design test and single factor test using the embryogenic callus of Ma7-6 and Quan172-1-1 cell lines of resistant P. massoniana . The situation of callus survival after cryopreservation was analyzed, and the maintenance of callus recovery of growth was evaluated. The optimal cryopreservation conditions for the embryogenic callus of nematode-resistant P. massoniana were screened out: pretreatment for 36-48 h, with 2.5%-10% DMSO, 0.4-0.5 mol/L sucrose and 15% PEG as cryoprotectant, and rewarming at 30 ℃-35 ℃. Under this condition, the embryogenic callus of the two lines had higher activity after rewarming, the rate of recovered embryogenic callus was also the highest (both the two cell lines were 100%), while the callus need less time to recovery (Ma7-6 need 23 d, Quan 172-1-1 need 15-19 d). Compared with the normal proliferation of callus, the callus after cryopreservation had no significant difference in appearance and microstructure. The result also showed that the callus after cryopreservation still had the ability to differentiate.