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The long-term preservation of forest genetic resources is a vital part of preserving our forest crops for future generations. Unfortunately, there are few genebanks dedicated to forest trees and very few methods for long-term preservation of forest genetic resources collections aside from field plantings of a limited number of seed-derived or elite clonal individuals. The use of cryopreservation for the long-term storage of elite germplasm is increasingly being used for the long-term preservation of clonal agronomic crops but for forest trees, such as Eucalyptus, the methodology for cryopreservation of diverse genetic resources collections has not been established. We report the successful cryopreservation of a germplasm collection of in vitro shoot cultures of thirteen Eucalyptus spp. lines consisting of two E. grandis x E. camaldulensis lines, seven E. urophylla x E. grandis lines, one E. grandis line, two E. grandis x E. urophylla lines, and one E. camaldulensis line. In a comparison of two cryopreservation methods, sucrose sensitivity limited the application of encapsulation-dehydration. However, with droplet-vitrification, all thirteen lines had good survival after cryopreservation in liquid nitrogen. A 30 min exposure to Plant Vitrification Solution 2 (PVS2) yielded post-liquid nitrogen survival between 38% and 85% depending on the line. One hundred shoot tips from all thirteen lines are currently in long-term storage as a germplasm collection.