People with multiple sclerosis (PwMS) who exhibit minimal to no disability are still over twice as likely to fall as the general population and many of these falls occur during walking. There is a need for more effective ways to detect preclinical walking balance deficits in PwMS. Therefore, the purpose of this study was to investigate the effects of optical flow perturbations applied using virtual reality on walking balance in PwMS compared to age-matched controls. We hypothesized that susceptibility to perturbations-especially those in the mediolateral direction-would be larger in PwMS compared to controls. Fourteen PwMS and fourteen age-matched controls walked on a treadmill while viewing a virtual hallway with and without optical flow perturbations in the mediolateral or anterior-posterior directions. We quantified foot placement kinematics, gait variability, lateral margin of stability and, in a separate session, performance on the standing sensory organization test (SOT). We found only modest differences between groups during normal, unperturbed walking. These differences were larger and more pervasive in the presence of mediolateral perturbations, evidenced by higher variability in step width, sacrum position, and margin of stability at heel-strike in PwMS than controls. PwMS also performed worse than controls on the SOT, and there was a modest correlation between step width variability during perturbed gait and SOT visual score. In conclusion, mediolateral optical flow perturbations revealed differences in walking balance in PwMS that went undetected during normal, unperturbed walking. Targeting this difference may be a promising approach to more effectively detect preclinical walking balance deficits in PwMS.