The metacestode of <jats:italic>Echinococcus multilocularis</jats:italic> is the etiological agent of alveolar echinococcosis. The metacestode stage used for research is maintained in rodents by serial passages. In order to determine whether cryopreservation of <jats:italic>E. multilocularis</jats:italic> metacestodes would be suitable for long-term maintenance and replace serial passages, isolates of different geographic origin were cryopreserved in 1984–1986. The aim of the current study was to test the viability of cryopreserved isolates following long-term cryopreservation (up to 35 years) and to determine the phylogenetic clades these isolates belonged to. Cryopreserved isolates were tested for viability <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic> in gerbils. <jats:italic>In vitro</jats:italic> results of 5 isolates indicated protoscolex survival in 13 of 17 experiments (76%) and metacestode survival in 5 of 12 (42%) <jats:italic>in vivo</jats:italic> experiments. <jats:italic>In vivo</jats:italic> results showed ‘abortive lesions’ in 13 of the 36 animals, 15 were negative and 8 harboured proliferating metacestode tissue containing protoscoleces. Genetic analysis confirmed the isolates belonged to European, Asian and North-American clades. In conclusion, the results of the current study indicate that metacestodes of <jats:italic>E. multilocularis</jats:italic> are able to survive long-term cryopreservation. Therefore, cryopreservation is a suitable method for long-term storage of <jats:italic>E. multilocularis</jats:italic> metacestode isolates and reduces the number of experimental animals.