In this study, the differences in terms of the fiber morphology and the chemical composition between the heartwood and sapwood of red gum (Eucalyptus camaldulensis Dehnh.), black willow (Salix nigra Marsh.), and oriental beech (Fagus orientalis Lipsky) were investigated. The results showed that the heartwood samples had shorter fibers and lower slenderness ratios than those of the sapwood samples. The differences in the vessel element length of the heartwood and sapwood of sampled tree species were statistically insignificant. The heartwood samples had less holocellulose and more klason lignin content. In addition, the ethanol, hot water, and cold water solubility values in heartwood samples were higher. The other morphological and chemical properties of the heartwood and sapwood depended on the tree species.