Scutellaria baicalensis is an important traditional Chinese medicine and Scutellaria flavonoids have received worldwide attention in recent years. It is the basis of controlling quality of S. baicalensis to develop a reliable genetic marker system used to identify locality of origin. Because of the characteristics of maternal inherited and high-rate of evolution, the cpDNA intergenic spacer can effectively elucidate the degree of genetic variation in different areas of the same species (populations), which can be used as the population-level DNA barcoding to locality identify. In this study, we have used the molecular phylogeography analysis for the three cpDNA intergenic spacers atpB-rbcL, trnL-trnF and psbA-trnH of 17 wild populations from different localities, which reveals the 20 haplotypes, including 13 polymorphic sites and constitutes a shallow gene tree. The authers have divided the haplotypes of S. baicalensis into three grades of population-level DNA barcoding according to the frequence and geographic distribution: 3 highest-frequency haplotypes as area-population-level DNA barcoding, 3 haplotypes were mainly shared by 2-3 adjacent populations as region-population-level DNA barcoding, and there are also 8 unique-population haplotypes as unique-population-level DNA barcoding. The result of this study reveals that population-level DNA barcoding is a reliable genetic marker used to locality identify of S. baicalensis.