The American Chestnut Foundation (ACF) has conducted a breeding program aimed at developing blight-resistant chestnut trees exhibiting the phenotype of American Chestnut [Castanea dentata (Marsh) Borkh]. Because such plants are difficult to propagate, we developed a protocol for in vitro multiplication of candidate blight-resistant plants resulting from the ACF breeding programs. Dormant shoots were taken from 5- to 8-year-old trees and forced, producing softwood growth for use as a source of explants for shoot multiplication. Best shoot proliferation took place on WPM containing 0.2 mg BA/L. Explant material for the rooting experiments was taken from 6- to 12-month-old proliferating cultures. The basal rooting medium consisted of WPM containing 0.01 mg IBA/L and was overlaid with a thin opaque layer. Rooting was enhanced overall with this bilayer approach. A “D/W” medium (DKW and WPM) was also used as a rooting medium containing 0.01 mg IBA/L and 0.2 mg BA/L, which further enhanced leaf quality and rooting for some genotypes. After several transfers on the bilayer system, explant growth appeared to become less juvenile in stem and leaf development and more analogous to mature later-season growth. The rooting responses and the time for rooting to be induced were highly variable among the different genotypes.