Eucalyptus species are currently one of the main feedstock for pulping and papermaking industry in China. In the present study, alkali-extractable hemicelluloses were isolated from different eucalyptus species (Eucalyptus camaldulensis, E. urophylla 3 grandis, and E. urophylla 3 E. tereticornis) at mild conditions prior to pulping. Structural characterization of these hemicellulosic polymers based on monosaccharide, molecular weight, Fourier transform infrared, 1 H, 13 C, and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance analysis revealed that these alkali-extractable polysaccharides shared the common structure composed of the (1!4)-linked-b-D-xylopyranosyl backbone with 4-O-methyl-a-D-glucuronic acid attached to O-2 of the xylose residues. The potential structures of the alkali-extractable hemicelluloses were proposed based on the comprehensive analysis. The well-characterized structures of these hemicelluloses could enlarge the industrial application of these hemicelluloses from the Eucalyptus species in a biorefinery process.