During mitosis, pairing of homologous chromosomes can be detrimental and has been correlated with gene misregulation, chromosomal aberrations, and various pathological diseases. We previously demonstrated that homologous chromosomes are spatially segregated, or antipaired, in neonatal human endothelial cells at metaphase/anaphase, which may help prevent abnormal recombination. However, it is unclear if this antipairing persists in adult endothelial cells. To test whether the antipairing, or one homolog per nuclear hemisphere motif, is conserved in adult endothelial cells, we examined human aortic endothelial cells at metaphase. Using ImmunoFISH and high-resolution confocal microscopy to visualize the chromosomes and centrosomes, we found that small homologous chromosomes 13, 15, 17, 19, 21, 22, and the sex chromosomes, XY, exhibit a loss of spatial segregation in human adult aortic endothelial cells. In contrast, fewer adult endothelial cells showed a loss of segregation for the larger chromosomes 1, 4, and XX, suggesting a gradual decline in the fidelity of spatial segregation of homologous chromosomes. Notably, we observed a higher frequency of abnormal pairing in both small and large chromosomes in adult aortic endothelial cells as compared to neonatal umbilical vein endothelial cells. These findings suggest that mechanisms governing chromosome antipairing may decline with aortic endothelial cell age, leading to increased susceptibility to abnormal pairing and cardiovascular disease.