The conservation of plant genetic resources is fundamental for the development of agriculture. The development of efficient cryopreservation protocols has become an effective tool to preserve cells, tissues and organs of different plant species. We sought here to develop an efficient method of cryopreservation of Eucalyptus grandis employing the V cryo-plate technique. This experiment examined the exposure of shoot tips to three different cryoprotectants (PVS2, PVS3, and PVS4). The cryoprotectants PVS2 and PVS4 proved to be more efficient among the cryoprotectants tested, as 44.4% of the shoot tips immersed in those solutions survived, and 31.1% generated new shoots. Ethylene glycol was found to be a key compound for the successful cryopreservation of shoot tips, in light of its low toxicity as compared to other cryoprotective compounds. Thus, the methodology developed here represents an effective method for the conservation of E. grandis germplasm through cryopreservation and the use of the V cryo-plate technique.