Gradient biomaterials have been developed and employed as an important tool in tissue engineering and biology research since the discovery that tissues and organs are non-homogeneous, exhibiting natural functional gradients in their structure or composition. Gradient biomaterials consist of relatively gradual continuous transitions in either compositional or mechanical properties. They have been used to study cellular responses such as cell adhesion, migration, proliferation, and differentiation, and may also be useful tools in drug discovery and development. Gradients made of hydrogels and nanofibers are widely used scaffolds in tissue engineering, which have aroused great interest owing to their tunable properties and analogy to the microenvironment of native tissues. In this chapter, we classify gradient biomaterials into two main cohorts, physical and chemical/biological gradients, and describe their features and applications, particularly as tooth or bone tissue scaffolds. © 2015 Elsevier Inc. All rights reserved.