American chestnut (Castanea dentata) is an endangered species in Canada. The population of American chestnut trees declined significantly due to the invasion of a fungal pathogen causing chestnut blight. American chestnut requires efficient strategies for propagation and preservation for species recovery. An in-vitro technology using liquid-based temporary immersion system (TIS) was developed for micropropagation of American chestnut. Optimum shoot growth was obtained in DKW (Driver and Kuniyuki Walnut) medium supplemented with 2.2 μM 6-benzylaminopurine and 1.0 μM gibberellic acid. Maximum microshoots developed roots on medium comprised 15.0 μM 3-Indolebutyric acid. A cryopreservation protocol was developed through droplet vitrification of shoots for germplasm preservation. Optimal regeneration of shoot tips was achieved by preculturing explants on stepwise concentrations of sucrose and subsequent dehydration in PVS3 for 30 min. These protocols may facilitate propagation and long-term preservation of American chestnut and possibly other woody plant species.