To accurately characterize branched polysaccharides with high molecular weights from medicinal and edible mushrooms and identify the limitations of size exclusion chromatography, molecular characteristics of polysaccharides from Tremella fuciformis were determined and compared by asymmetrical flow field-flow fractionation coupled with multiangle laser light scattering and refractive index detection, and size exclusion chromatography coupled with multiangle laser light scattering and refractive index detection, respectively. Results showed that molecular weights of three batches of T. fuciformis polysaccharides were determined as 2.167 x 10(6) (TF1), 2.334 x 10(6) (TF2), and 2.435 x 10(6) Da (TF3) by size exclusion chromatography, and 3.432 x 10(6) (TF1), 3.739 x 10(6) (TF2), and 3.742 x 10(6) Da (TF3) by asymmetrical flow field-flow fractionation, as well as 3.469 x 10(6) Da (TF1) by off-line multiangle laser light scattering, respectively. Results suggested that size exclusion chromatography was unable to accurately characterize T. fuciformis polysaccharides, which may be due to its limitations such as shear degradation and abnormal coelution. Compared to size exclusion chromatography, asymmetrical flow field-flow fractionation could be a better technique for the molecular characterization of branched polysaccharides with high molecular weights from medicinal and edible mushrooms, as well as from other natural resources.