Abstract: On the basis of cloning and expression analysis of CCoAOMT and SAMS genes related to 2-methoxy-1,4-naphthoquinone (MNQ) synthesis of Impatiens hawkeri, the molecular mechanism of its MNQ synthesis was discussed, which provided theoretical basis for MNQ synthesis of Impatiens. In this study, based on the determination of MNQ content in I. hawkeri, the target genes were screened by transcriptome sequencing, and 1 CCoAOMT and 2 SAMS genes of I. hawkeri were cloned, named IhCCoAOMT, IhSAMS1 and IhSAMS2 respectively, whose expression patterns were analyzed. The results showed that all three treatments could promote the accumulation of MNQ, and dark culture had the best effect. The cDNA of IhCCoAOMT was 729 bp and encoded 242 aa, its protein has AdoMet_MTases superfamily domain, and the cDNAs of IhSAMS1 and IhSAMS2 were 1179 bp and 1173 bp, encoded 393 aa and 391 aa, respectively, and their proteins have S-AdoMet_synt superfamily domain. Phylogenetic analysis showed that IhCCoAOMT gene of I. hawkeri was most closely related to I. glandulifera. IhSAMS1 and IhSAMS2 of I. hawkeri may be paralogous genes. The qRT-PCR analysis showed that the three genes were expressed in three treatments and four culture periods of I. hawkeri, in which the expression of IhCCoAOMT gradually increased in dark culture and 1,4-dihydroxy-2-naphthoate (DHNA) treatment, and the expression was highest on the 60th day of dark culture. The expression of IhSAMS1 and IhSAMS2 were gradually downregulated in dark culture and DHNA treatment, but the expression level of IhSAMS1 was higher in DHNA treatment, and the expression level of IhSAMS2 was higher in dark culture and the overall expression level was higher. In conclusion, it’s speculated that IhCCoAOMT played a key role in the late stage of MNQ treatment, while IhSAMS1 and IhSAMS2 played an important role in the early stage of MNQ treatment , and IhSAMS2 was more significant.