The genus Scutellaria L., Lamiaceae family, Scutellarioideae sub-family is known as the most important medicinal plant in the world. This research aimed to investigate the flavonoid content of seven Scutellaria species from the center, southwest, and west of Iran. Via thin layer and column chromatography, the flavonoid was extracted from leaves and then purified. To screen the flavonoid compositions, a chromatographic method was applied by liquid chromatography mass spectrometry on a triple quadrupole mass spectrometer (LC/TQMS/MS). Fingerprint analysis was implemented so as to characterize a total of 73 chemical compounds, from which 71 compounds were flavonoids belonging to different classes. Flavone with 37 derivatives possessed major values. The most abundant flavonoid compounds were observed in S. multicaulis (23 compounds) and S. patonii (22 compounds). Flavonoid composition, including apigenin, kaempferol, quercetin, and hydroxylflavones represented remarkable derivatives. A total of 45 flavonoids, one tannin and one anthraquinone compound, were observed to be primarily separated and identified for Scutellaria species. Moreover, six categorized chemical groups were identified in this genus and proposed as chemical barcodes. The specific chemical groups strongly provided the boundaries of Scutellaria species, the pharmacological value enhancement, breeding programs, and comprehensive documents of the species. According to the results, LC/TQMS/MS was proven a dominant method regarding genus Scutellaria.