Scutellaria baicalensisis a well-studied medicinal plant belonging to the Lamiaceae family, prized for the unique 4′-deoxyflavones produced in its roots. In this study, three native species to the Americas,S. lateriflora,S. arenicola,andS. integrifoliawere identified by DNA barcoding, and phylogenetic relationships were established with other economically important Lamiaceae members. Furthermore, flavone profiles of native species were explored. 4′-deoxyflavones including baicalein, baicalin, wogonin, wogonoside, chrysin and 4′-hydroxyflavones, scutellarein, scutellarin, and apigenin, were quantified from leaves, stems, and roots. Qualitative, and quantitative differences were identified in their flavone profiles along with characteristic tissue-specific accumulation. 4′-deoxyflavones accumulated in relatively high concentrations in root tissues compared to aerial tissues in all species exceptS. lateriflora. Baicalin, the most abundant 4′-deoxyflavone detected, was localized in the roots ofS. baicalensisand leaves ofS. lateriflora, indicating differential accumulation patterns between the species.S. arenicolaandS. integrifoliaare phylogenetically closely related with similar flavone profiles and distribution patterns. Additionally, theS. arenicolaleaf flavone profile was dominated by two major unknown peaks, identified using LC–MS/MS to most likely be luteolin-7-O-glucuronide and 5,7,2′-trihydroxy-6-methoxyflavone 7-O-glucuronide. Collectively, results presented in this study suggest an evolutionary divergence of flavonoid metabolic pathway in theScutellariagenus of Lamiaceae.