The dried rhizomes of Coptis chinensis have been extensively used in heat clearing,dampness drying, fire draining, and detoxification by virtue of their major bioactivecomponents, benzylisoquinoline alkaloids (BIAs). However, C. teeta and C. chinensisare occasionally interchanged, and current understanding of the molecular basis of BIAbiosynthesis in these two species is limited. Here, berberine, coptisine, jatrorrhizine,and palmatine were detected in two species, and showed the highest contentsin the roots, while epiberberine were found only in C. chinensis. Comprehensivetranscriptome analysis of the roots and leaves of C. teeta and C. chinensis,respectively, identified 53 and 52 unigenes encoding enzymes potentially involved in BIAbiosynthesis. By integrating probable biosynthetic pathways for BIAs, the jatrorrhizinebiosynthesis ill-informed previously was further characterized. Two genes encodingnorcoclaurine/norlaudanosoline 6-O-methyltransferases (Cc6OMT1 and Cc6OMT2)and one gene encoding norcoclaurine-7OMT (Ct7OMT) catalyzed enzymatically O-methylate (S)-norcoclaurine at C6 that yield (S)-coclaurine, along with a smalleramount of O-methylation occurred at C7, thereby forming its isomer (isococlaurine).In addition, scoulerine 9-OMT (CtSOMT) was determined to show strict substratespecificity, targeting (S)-scoulerine to yield (S)-tetrahydrocolumbamine. Taken together,the integration of the transcriptome and enzyme activity assays further provides newinsight into molecular mechanisms underlying BIA biosynthesis in plants and identifiescandidate genes for the study of synthetic biology in microorganisms.