Double-stranded (ds) DNA from salmon testes has been incorporated into PVA hydrogels obtained by a technique of repeated freezing and thawing. The cryogels obtained are free of potential toxic species like chemical cross-linkers, and consequently, they can be used in pharmaceutical or medical applications. These cryogels show a good mechanical resistance and a white and opaque appearance caused by a heterogeneous porous structure. Encapsulated DNA molecules can be in a compacted or an extended conformation in the PVA matrix and can be controlled by tailoring the degree of crystallinity of the PVA network; this is supported by fluorescence microscopy and UV and FTIR spectroscopic studies. The two forms of encapsulated DNA were observed for different types of matrixes: an extended one in a more crystalline network and a globular one in a more amorphous one. Different associations of base pairs have also been observed. PVA cryogel crystallinity could be tailored by the cryogel contact with different salt solutions. Cryogel surface (scanning electron microscopy) and bulk morphology (porosimetry), swelling, DNA retention, and delivery kinetics have also been studied. All these investigations clearly show strong interactions between PVA and DNA.