This study examined time-sequential changes in the biomechanical and morphological properties of articular cartilage that had received cryopreserved osteochondral allografting. Osteochondral blocks obtained from the femurs of 18 rabbits were cryopreserved with dimethylsulfoxide (DMSO), using a two-step freezing method, and allografted to the femurs of another 18 rabbits. Specimens for biomechanical and morphological examinations were prepared at the second, fourth, and twelfth weeks after allografting (n = 18). In 12 allografted rabbits, biomechanical features were examined with an indentation test apparatus, and histological changes were studied with a light microscope (second week, n = 4; fourth week, n = 4; twelfth week, n = 4). In the other 6 allografted rabbits, cartilage surfaces were studied with a scanning electron microscope (second week, n = 2; fourth week, n = 2; twelfth week, n = 2). For controls, fresh, DMSO-treated, or DMSO-treated + cryopreserved specimens were examined biomechanically and morphologically. In the time-sequential examination of biomechanical features, both the parameter for elasticity (i.e., ratio of instant elastic strain to maximum strain) and the parameter for viscosity (i.e., average retardation time) significantly changed. Light microscopy showed chronological decreases in safranin-O staining intensity in the matrix, and progression of degeneration. On scanning electron microscopy, disruption of the cartilage surface was also recognized. Therefore, changes in biomechanical properties due to cryopreservation could cause irreversible changes in the cartilage in cryopreserved osteochondral allografting.