Abstract: Root flavonoids of 5 Scirpus L. species: Cypereae Colla. Tribe, Cyperaideae Kostel. subfamily and Cyperaceae family (S. holoschenus L., S. lacustris L., S. littoralis Kuntze, S. maritimus L. and S. multicaule) from different parts of Markazi Province, Iran area were studied using 2-dimentional Paper Chromatography (2-DPC) and Thin Layer Chromatography (TLC). Flavonoids are as one set of the polyphenolic compounds among secondary metabolites in different organs of plants that are used in chemotaxonomy. Also many flavonoids are active principles of medicinal plants, exhibit pharmacological effects and contribute to human health. Voucher samples were prepared for reference as herbarium vouchers. Results showed all of studied taxa contain flavonoid sulphates, flavone C and C-/O-glycosides and aglycones in their roots while Rutin, Myricetin and Vitexin were just found in S. maritimus. Also presence of Morin, Tricin and Loteulin in all of the species roots with the exception of S. maritimus are more valuable tools for taxa separation. Kaempferol was found in S. lacustris and S. littoralis species, where as others lack.