Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7–63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved.