In this work, we shed new light on highly discussed chromatin fragmentation of cryopreserved cells. Moreover, for the first time, we described replicating cell-specific DNA damage and higher-order chromatin alterations after freezing/thawing. We identified DNA structural changes associated with the freeze/thaw process and correlated them with the viability of cells that had been frozen and thawed. We simultaneously evaluated DNA defects and higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze/thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing and accompanied by the shrinkage of their nuclear envelopes, had the highest rate of survival upon freezing. The results of our work will facilitate the future design of compounds and procedures [1] to decrease injury to cryopreserved cells.