In this study, morphological characters, chemotaxonomy and the flavonoid compounds of five Marrubium species are investigated. To this end, 57 accessions of five Marrubium species were collected from their natural habitats in the Zagros areas of Iran. In order to conduct morphological studies, 18 qualitative and 11 quantitative characters were surveyed. In chemotaxonomy studies, the leaf flavonoids of each species were extracted by crude MeOH and were subjected to thin-layer chromatography with silica gel 60F. The extracted flavonoids were purified by column chromatography and UV spectra (200–400 nm). Cluster analysis with Euclidean distance and SPSS v.20 was applied to investigate the taxonomic status of each species using the presence and absence of color spots in thin-layer chromatography and morphological characters. Based on our results, a total of 51 flavonoid compounds were identified in Marrubium species. The majority of substitution patterns were also identified as hydroxylation, methoxylation and glucosylation. The cluster analysis of data from both flavonoids and morphology showed that Marrubium cuneatum and Marrubium vulgare comprise a complex group. The highest flavonoid diversity was observed in Marrubium anisodon and Marrubium vulgare. In addition, Marrubium cuneatum, Marrubium vulgare and Marrubium anisodon demonstrate the highest morphological diversity. Morphologically, the characters such as leaf form, bracteole indumentums, number and form of calyx teeth and bracteole length were useful to discriminate Marrubium species. Finally, morphological characters and flavonoid profiles were found to be appropriate markers to detect the variation and taxonomic status in this genus.