Substrate stiffness is known to impact characteristics including cell differentiation, proliferation, migration and apoptosis. Hydrogels are polymeric materials distinguished by high water content and diverse physical properties. Gradient stiffness hydrogels are designed by the need to develop biologically friendly materials as extracellular matrix (ECM) alternatives to replace the separated and narrow-ranged hydrogel substrates. Important new discoveries in cell behaviors have been realized with model gradient stiffness hydrogel systems from the two-dimensional (2D) to three-dimensional (3D) scale. Basic and clinical applications for gradient stiffness hydrogels in tissue engineering and regenerative medicine continue to drive the development of stiffness and structure varied hydrogels. Given the importance of gradient stiffness hydrogels in basic research and biomedical applications, there is a clear need for systems for gradient stiffness hydrogel design strategies and their applications. This review will highlight past work in the field of gradient stiffness hydrogels fabrication methods, mechanical property test, applications as well as areas for future study. (c) 2017 Wiley Periodicals, Inc.