This study was designed to evaluate the effect of cryopreservation on the glycosaminoglycan (GAG) content of the aortic allografts. Twenty-one porcine aortic valves were obtained. Five aortic roots were immediately analyzed without cryopreservation, eight were cryopreserved in closed leaflet position, and eight in open leaflet position. The groups were compared in terms of GAG concentration and subclass proportion in three different zones including the aortic root wall, the commissures, and the leaflets. GAG content at the commissures was significantly lower in the closed leaflet group than in the other groups (P = 0.001). The electrophoretic analysis did not show any significant difference in the zonal distribution of GAG classes between groups. Quantitative analysis in various aortic valve zones suggests that cryopreservation can alter the GAG content. Cryopreservation of the aortic valve in an open leaflet position can preserve the matrix more efficiently and might prolong the durability of the aortic allograft.