Peach (Prunus persica (L.)) is one of the most important vegetatively propagated crop germplasm resources in China. Cryopreservation has been implemented in genebanks worldwide to conserve vegetatively propagated plant collections by using shoot tips, pollen or dormant buds. In this study, the effects of moisture content, rehydration treatment, and storage temperature (4 ℃ and cryopreservation) on in vitro germination levels of pollen after storage were investigated for 15 peach cultivars. Results showed that: the moisture content for peach pollen cryopreservation were determined; The optimal rehydration condition was found to be cultivar-dependent; In vitro germination levels of peach pollen after cryopreservation were as high as 80%; After 4 years storage, in vitro germination levels of peach pollen after cryopreservation (in vitro germination levels were higher than 30% for 14 cultivars) significantly higher than those were stored under 4 ℃ low temperature (in vitro germination levels of 0). And in vitro germination levels of pollen for 11 cultivars after 4 years cryopreservation were not changed significantly, even significantly higher than the in vitro germination levels before storage. In conclusion, this study will benefit for the establishment of pollen large scale cryopreservation for the new National Crop Genebank of China in future.