Marrubium and Ballota are known to be important medicinal plants belonging to the Lamiaceae family. Their aerial parts have been widely used in traditional medicine. The present study, for the first time, aimed to investigate flavonoid constituents and chromatographic pattern of the methanolic extract of leaf from Marrubium and Ballota species. The technique was performed on High Performance Liquid Chromatography (HPLC)-Micromass Quattro micro Atmospheric Pressure Ionization (API) Mass Spectrometer in six taxa. A total of 59 chemical compounds were recognized, among which 49 flavonoids, three polyphenols, and one methoxyphenol were identified. In addition, five chemical groups were recognized in Marrubium and Ballota species. It is noteworthy that Marrubium and Ballota species provide a major source of apigenin, kaempferol, and quercetin glycosides. The flavonoid compounds, such as licoricidin, sophoraflavanone G, and methyl robustone were highly frequent considering qualitative markers for the genus Marrubium. Despite the striking similarity between Marrubium and Ballota species, they were accurately separated using chemical markers, particularly the mass (MS/MS) spectra of flavonoid compounds, which can develop the functional products and pharmaceutical, chemotaxonomic, and chemo diversity purposes for Marrubium and Ballota genera.