Qualitative and quantitative analysis of non-starch polysaccharide (NSP) from Pseudostellaria heterophylla in China (Guizhou, Anhui and Fujian) were performed using high performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID), saccharide mapping based on polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) and high performance thin layer chromatography (HPTLC). Results showed that PACE and HPTLC fingerprints of enzymatic hydrolysates of NSP were similar. Moreover, the results indicated that 1,4-beta-d-Galp, 1,5-alpha-Araf, 1,4-alpha-d-GalAp and 1,4-beta-d-Glcp linkages existed in NSP. Based on the results of HPSEC-MALLS/RI, the Mw, Rg, and contents of NSP in P. heterophylla were ranging from 4.37x10(4) to 1.70x10(5) Da, 46.4 to 65.9 nm, and 0.43% to 1.41%, respectively. There were no significant difference (p > 0.05) observed among the three main producing areas, which suggested that polysaccharides could be used as marker for quality control of P. heterophylla.