Cryopreservation is an essential step for utilizing various cell types for biological research and medical purposes. At the same time, there is a lack of data on the effect of cryopreservation, especially when prolonged, on the karyotype of cells. In the present work, we analyzed the genetic stability of cells subjected to a cryopreservation procedure. The objects were immortalized Chinese hamster lung fibroblasts (CHL V-79 RJK line) and human endometrial mesenchymal stem/stromal cells (eMSCs). We showed that short-term cryopreservation in liquid nitrogen for up to 6 months did not affect the karyotype stability of CHL V-79 RJK and eMSCs. On the contrary, karyotyping of G-banded metaphase chromosomes in cells underwent 10-year cryopreservation, which revealed genomic instability in both cell lines associated with the variability of chromosome number in cells, random chromosomal rearrangements, and condensation disorder in homologs. In addition, we found out that long-term cryopreservation of eMSCs does not affect the expression of their typical surface markers and morphology, but results in a significant reduction in proliferative potential and early manifestation of cellular senescence features upon eMSCs culturing. Thus, we concluded that the long-term cryopreservation of cells of different types and biological origin can lead to irreversible changes of their karyotype and acceleration of cellular senescence.