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In traditional Chinese medicine research, seaweeds used for drug and health food development mainly refer to Sargassum pallidum (Turn.) C. Ag. (SP) and Sargassum fusiforme (Harv.) Setch. (SF). In the Chinese Pharmacopoeia, polysaccharides are the main quality control indicators. The total sugar content is determined by the anthrone–sulfuric acid method to evaluate the quality of seaweeds. However, this method cannot reflect the structural characteristics of seaweed polysaccharides and cannot distinguish among different seaweed varieties. Given this, to comprehensively evaluate the quality of different seaweeds, this study conducted a thorough analysis of the primary structure of polysaccharides in 11 batches of SF and 7 batches of SP. The neutral sugar content was determined by the anthrone–sulfuric acid colorimetric method, weight average molecular weight (Mw) by high‐performance size exclusion chromatography coupled with multi‐angle laser light scattering and refractive index detector method, sulfate content by ion chromatography and monosaccharide composition by ultrahigh‐performance liquid chromatography method, while also using professional software for statistical and similarity analysis. The results showed significant differences (p < 0.05) in neutral sugar content and Mw between S. fusiforme polysaccharides and S. pallidum polysaccharides, which can be used to distinguish SF and SP. The monosaccharide composition fingerprint was analyzed using chemometric methods, and it was found that fucose and glucose could serve as differential markers to distinguish SF and SP. This study further deepens the understanding of polysaccharides in seaweeds and more comprehensively evaluates the quality of different seaweeds. [ABSTRACT FROM AUTHOR]