Abstract: The flavanone-3-hydroxylase（F3 H） gene was cloned from the flower of Meconopsis wilsonii by rapid amplification of cDNA ends（RACE） and named MwF3 H. Its physical and chemical properties, protein domain, evolutionary relationship and gene expression level were analyzed to explore the expression pattern of MwF3 H gene in different flowering stages of M. wilsonii. The results showed that the length of the coding region of the gene was 1 104 bp, encoding 367 amino acids, the molecular weight of the protein is 41.444 kDa, and the theoretical isoelectric point（pI） is 4.98, belonging to the PLNO2515 supergene family. The construction of a phylogenetic tree found that M. wilsonii and M. zangnanensis is closely related, and the same species is different in different geographical areas. The qPCR results showed that the MwF3 H gene is expressed to varying degrees in different stages of the development of M. wilsonii, except for the bud coloring stage, the highest expression level is in the full bloom stage. The results showed that MwF3 H gene most probably was involved in the biosynthesis of related pigments during M. wilsonii flowering. It may play a key role in the process of flower color formation.