•Cotyledons were used as explants in the rapid shoot regeneration method in hemp.•TDZ applied in MS medium were found to be capable of inducing in vitro shoots from cotyledons superior than BA and ZT.•Younger cotyledons (2–3 DAP) produced significantly higher regeneration frequency than elder ones (5–6 DAP).•The regeneration method was suitable while partly genotype dependent.•The method may afford an alternative method for micropropagation and germplasm conservation, and the in vitro plantlets may be suitable to set up a transformation system. Hemp (Cannabis sativa) is an annual multipurpose crop that is distributed worldwide. An efficient regeneration protocol is needed for hemp genetic transformation, micropropagation, and germplasm conservation. We describe here a rapid protocol for in vitro shoot regeneration that uses cotyledons as explants. We concluded that TDZ in MS medium is more efficient in inducing in vitro shoots from cotyledons than BA and ZT. The best result, 51.7% induction frequency and 3.0 shoots per shoot explant, was recorded in MS medium containing 0.4mgl−1 TDZ and 0.2mgl−1 NAA (T4N2). The in vitro shoots grew to the height of 1.5–2cm in 3–4 weeks after culture initiation. At that time, approximately 80% of the shoots were rooted well in half-strength Ms medium combined with 0.5–2mgl−1 IBA for 4–5 weeks before acclimation. It was observed that younger cotyledons (2–3 days after planting (DAP) were more useful as explants than older ones (5–6 DAP) because they had a significantly higher regeneration frequency. We cultured 3 DAP cotyledons of eight cultivars in T4N2 medium to test the efficiency. The regeneration frequency varied from 35.7% to 54.8%, which indicated that this regeneration protocol was effective, although partly genotype dependent. We regard this protocol as an alternative method for micropropagation and germplasm conservation, and the in vitro plantlets may be suitable to set up a transformation system.