Abstract: In order to screen the stable reference genes for quantity real-time PCR suitable in hemp seed germination and osmotic stress conditions, 10 candidate reference genes (CDPK, eIF4E, TIP41, UBQ, EF1α, PP2A, Actin, Clathrin, TUB, DHS2) were analysed in this study. ‘Yunma 7’ seeds were treated with distilled water (normal germination) and 20% PEG (osmotic stress), respectively. Seedling samples were taken at 3 different seed germination periods (3 d, 5 d and 7 d after treatment). The expression of candidate reference genes was detected by qRT-PCR technology, and the expression stability was evaluated by GeNorm, Normfinder and BestKeeper software packages. The appropriate reference genes were obtained by comprehensive analysis of RefFinder online software. Among 10 candidate reference genes, the stability of PP2A gene was the best in the normal germination stage of hempseed, the stability of UBQ gene was the best in seed germination under osmotic stress, and the expression of eIF4E (as a reference gene) was the most stable under these two conditions. In this study, the most suitable reference genes for qRT-PCR analysis of hempseed germination under osmotic stress were determined, which laid a foundation for the expression analysis of genes related to seed germination under osmotic stress of hemp in the future.