Background: The aim of the present study was to examine the composition and content of the polysaccharide complex and polysaccharide fractions from yellow iris (Iris pseudacorus L.) rhizomes growing in Russian Federation (Moscow region). Materials and Methods: Traditional pharmacognostic methods were used in the analysis of polysaccharides as biologically active substances. The total amount of polysaccharides was determined by a gravimetric procedure with alcohol precipitation. Fractions of polysaccharides were isolated by sequential changing of solvents. The content of monosaccharides was determined by the method of high-performance liquid chromatography with refractometric detection (HPLC-RID) after acid hydrolysis. HPLC-RID procedure was performed using Chromatograph Agilent 1260 Infinity, Sugar-Pak column (300×6.5 mm), and isocratic elution mode. Results: Polysaccharide complex was isolated, the total content of polysaccharides in the rhizomes of I. pseudacorus was determined, as well as their distribution by fractions (water-soluble polysaccharides, pectic substances, hemicellulose A, hemicellulose B), the composition of individual sugars (glucose, xylose, galactose, mannose, rhamnose, arabinose) was determined for each fraction. Conclusion: In our experiment polysaccharide complex was first identified and described in detail for I. pseudacorus grown in Moscow region.