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Abstract Background The genomic stability of stem cells to be used in cell therapy and other clinical applications is absolutely critical. In this regard, the relationship between in vitro expansion and the chromosomal instability (CIN), especially in human amniotic fluid cells (hAFCs) has not yet been completely elucidated. Objective To investigate the CIN of hAFCs in primary and long-term cultures and two different culture mediums. Materials and Methods After completing prenatal genetic diagnoses (PND) using karyotype technique and chromosomal analysis, a total of 15 samples of hAFCs from 650 samples were randomly selected and cultured in two different mediums as AmnioMAX II and DMEM. Then, proliferative cells were fixed on the slide to be used in standard chromosome G-banding analysis. Also, the senescent cells were screened for aneuploidy considering 8 chromosomes by FISH technique using two probe sets including PID I (X-13-18-21) & PID II (Y-15-16-22). Results Karyotype and interphase fluorescence in situ hybridization (iFISH) results from 650 patients who were referred for prenatal genetic diagnosis showed that only 6 out of them had culture- derived CIN as polyploidy, including mosaic diploid-triploid and diploid-tetraploid. Moreover, the investigation of aneuploidies in senesced hAFCs demonstrated the rate of total chromosomal abnormalities as 4.3% and 9.9% in AmnioMAX- and DMEM-cultured hAFCs, respectively. Conclusion hAFCs showed a low rate of CIN in two AmnioMAX II and DMEM mediums and also in the proliferative and senescent phases. Therefore, they could be considered as an attractive stem cell source with therapeutic potential in regenerative medicine.