The molecular characterization and rheological properties of hemicellulose B (Hemi. B) fractions isolated from sorghum bran (SBR), sorghum bagasse (SBA) and sorghum biomass (SBI) were undertaken. The monosaccharide analysis results confirmed that these polysaccharides had a typical arabinoxylan structure. High performance size exclusion chromatography study indicated that Hemi. B from SBR and SBA had a higher weight average molar mass (Mw) (383–397 kDa), radius of gyration (Rgz) (34.7–34.9 nm), intrinsic viscosities (ηw) (1.25–1.29 dL/g) and lower polydispersity index (Mw/Mn) (2.76–2.79) than Hemi. B from SBI. The Mw, Rgz, ηw and Mw/Mn of the Hemi. B from SBI were 261 kDa, 31.6, 0.931 and 6.01 respectively. The Hemi. B from SBR and SBA had a similar random coil shape, while the Hemi. B from SBI was more like a compact sphere in shape. The Hemi. B isolated from SBR showed a typical shear thinning behavior at all concentrations. But the Hemi. B from other two sources (SBA and SBI) exhibited a shear shinning behavior at high concentration, showing Newtonian behavior at lower concentration. The critical concentration was also calculated to evaluate the molecular properties. The lower Mw of Hemi. B shows a highest value of critical concentration (c∗), which was in coordinate with the negative correlation between c∗ values and Mw of different arabinoxylan varieties.