Abstract: As a traditional Chinese medicine, Scutellaria barbata D. Don has an important economic value. However, little molecular-based study was reported that limits the utilization and development of the resource. In this study, Illumina HiSeq X Ten was used to sequence the transcriptomes of S. barbata to obtain 125 823 unigenes assembled by the de novo DNA synthesis. The average length of N50 was 826 bp averaging 586 bp with 74 185 unigenes annotated. There were 14 832 differentially expressed unigenes in the above- and under-ground parts of the plants, of which 6 941 were up-regulated and 7 891 down-regulated. According to the KEGG database, 89 unigenes related to the flavonoid biosynthesis pathway. Among them 36 were differentially expressed. By comparing the genes in the above- and the under-ground parts, it was found the important enzymes associated with the flavonoid synthesis to be significantly down-regulated in the parts grown below the ground level. In other words, the stems and leaves were the major parts on a plant that contained the medicinally active components.