Belonging to Lamiaceae family, the genus Teucrium L. is a rich source of secondary metabolites used in pharmacology and industrial medicine. This study aimed to investigate the flavonoid contents of methanol extract and the chemical markers of Teucrium at specific and infraspecific levels and identify the chemotypes of its accessions. Nineteen accessions, eight species, and five subspecies were collected from three sections (Teucris, Polium, and Scordium) belonging to the natural habitats in the west, southwest, centre, and south of Iran. Total flavonoid extraction was attained by the air-dried leaf of Teucrium species and 90% methanol. HPLC coupled with triple quadrupole mass analyser was applied to screen and identify the chemical constituents. Multivariate analyses were implemented by cluster and principal component analysis. Chemical investigation specified 148 chemical compounds, of which 135 flavonoids were tentatively identified. The most abundant derivatives belonged to flavones (41 derivatives), flavonols (37 derivatives), and isoflavonoids (22 derivatives). Based on the cluster analysis, 13 groups of chemical markers were formed in sections Teucris and Scordium and five groups in section Polium as chemotaxonomic indicators. The specified chemotypes were represented by ten in the sections Teucris and Scordium and by six in the section Polium. A relationship was suggested between type and flavonoid quantity (number), altitude, and geographical location. The present study provided valuable information for pharmacological research, bioactivity assessment, and chemotaxonomic purposes. This is the first report on the presence of chemotypes, chemical markers, and quality assessments for the genus Teucrium.