Culture conditions play an important role in regulating the genomic integrity of HPSCs. We report that HPSCs cultured in Essential 8 (E8) and mTeSR, two widely used media for off-feeder culturing of HPSCs, had many fold higher levels of ROS and higher mitochondrial potential than cells cultured in KSR containing media. HPSCs also exhibited increased levels of 8-hydroxyguanosine, phospho-histone-H2A.X and p53, as well as increased sensitivity to γ-irradiation in these two media. HPSCs in E8 and mTeSR had increased incidence of alterations in their DNA sequence, reflecting genotoxic stress, in addition to changes in nucleolar morphology and number. Supplementing E8 and mTeSR with antioxidants provided only a partial rescue. Our results suggest that it is essential to determine cellular ROS levels in designing culture media as it affects the genomic integrity of HPSCs and will limit their use in studying development and in regenerative medicine.