OBJECTIVE: We aimed to explore the cross-sectional relation of unilateral knee pain severity and temporal asymmetry during walking and to determine relations of temporal asymmetry during walking to 2-year changes in ipsilateral and contralateral knee pain in those with mild-to-moderate unilateral knee pain. METHODS: The Multicenter Osteoarthritis Study is a prospective cohort study of adults with or at risk for knee osteoarthritis. The current study included participants with unilateral knee pain. Gait was assessed during self-selected and fast walking at baseline. Knee pain was assessed at baseline and 2 years. We calculated limb symmetry indices (LSIs; nonpainful limb/painful limb × 100) for stance, single-limb support time, and double-limb support time, then examined their relations to unilateral knee pain severity, incident contralateral knee pain, and persistent ipsilateral knee pain. RESULTS: Unilateral knee pain severity was not associated with temporal asymmetry during self-selected or fast walking. At 2 years, 17.1% of participants had incident contralateral knee pain and 51.4% had persistent ipsilateral knee pain. For self-selected walking, greater LSIs (i.e., longer time on the nonpainful limb) for stance and single-limb support time were associated with decreased odds of incident contralateral knee pain. Measures of temporal asymmetry were not associated with persistent ipsilateral knee pain, except for single-limb support time during fast walking. CONCLUSION: For those with unilateral knee pain, temporal asymmetry during walking is not associated with pain severity. However, select measures of stance and single-limb support time during self-selected and fast walking relate to longitudinal knee pain outcomes.