Rosa chinensis var. spontanea predominantly emits 1,3,5-trimethoxybenzene together with methyleugenol and isomethyleugenol as minor floral scent compounds. Two O-methyltransferases (OMTs), designated as RcOMT1 and RcOMT2, were isolated from rose flower petals using homology-based screening strategies. RcOMT1 efficiently methylated eugenol and isoeugenol to yield volatile methyleugenol and isomethyleugenol, respectively. Furthermore, the mRNA transcripts of RcOMT1 were highly expressed in floral organs and the expression pattern coincided with intracellular content changes of methyleugenol and isomethyleugenol in rose flowers. In contrast, RcOMT2, which shows 94% similarity with caffeic acid O-methyltransferase (COMT) of Prunus amygdalus, was expressed in all tissues tested and had the highest activity with caffeic acid, a typical substrate for COMT. However, this COMT-like OMT also showed some degrees of activity with all three putative precursors of 1,3,5-trimethoxybenzene.