Abstract: UDP-rhamnose is a rhamnose donor in a reaction catalyzed by UDP-rhamnose synthase (RHM), and plays an important role in the biosynthesis of rhamnoside compounds. The current literature suggests that there are only a few genes can encode the corresponding enzymes to participate in UDP-rhamnose biosynthesis in plants. In this study, two RHM genes (FmRHM1 & 2) were first cloned by using the transcriptomic data of Fallopia multiflora (Thunb) Harald and the multidimensional analysis, including bioinformatics, functional identification in vitro and tissue-specific expression analysis. The results showed that the open reading frame (ORF) of FmRHM1 & 2 genes both were 2 013 bp, encode proteins consisting of 670 amino acids with a calculated molecular mass of 75.6 kDa, and the theoretical isoelectric points of 6.20 and 7.19, respectively. Bioinformatic analysis also indicated that FmRHM1 & 2 contained 2 special sequences of GxxGxxG/A and YxxxK. The phylogenetic analysis showed that the FmRHM gene has a high homology with RHM of other species. The results of enzyme activity in vitro revealed that both recombinant FmRHM1 and FmRHM2 have catalytic activities for converting UDP-glucose into UDP-rhamnose. Measurements of tissue-specific expressions showed that the expression levels of FmRHM1 and FmRHM2 were lower in roots. On the contrary, the 2 genes showed significantly high expression in the stems and leaves. In conclusion, we have cloned and characterized the RHM gene function for the first time in F. multiflora. Here we have provided the preliminary data suggesting the need for further research on UDP-rhamnose biosynthesis by microorganisms.