Biodiversity of threatened species can be preserved in cryopreservation repositories, allowing storage of biological information and future re-introduction to the wild. The success of ex situ programs for species conservation relies on using simple and efficient cryopreservation techniques. Bryophytes may be classified as stress-tolerant plants, but a large fraction worldwide is nevertheless threatened by extinction to various degrees. The aims of this study were to investigate how to exploit natural stress tolerance of bryophytes in the development of simple cryopreservation protocols without the use of cryoprotectants or pretreatment. We studied regeneration capacity of bryophytes found at different moisture and light conditions in vivo (reflecting varying levels of plant desiccation tolerance), both sampled from herbarium specimens of varying ages and also collected in situ during winter season and thus naturally cold acclimated. Furthermore, to identify optimal samples for direct cryopreservation we investigated how the ability to regenerate corresponds to the fragment size. We show that young samples (≤ 7 yrs) of the most desiccation tolerant species regenerate via protonemata, where shoot tips regenerate better than small plant fragments. Herbaria can therefore represent a source of samples for direct cryopreservation when using desiccation tolerant species, stored for a limited time. Conversely, all species sampled in situ regenerated, mainly through budding. In these plants, the number of regenerated samples is positively correlated to desiccation tolerance, and they show a higher frost tolerance than previously reported. Cryopreservation without pretreatment or cryoprotectants of cold acclimated bryophytes can therefore be used as a simple and efficient method for storage of bryophyte tissue.