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Gait variability is a marker of gait performance and future mobility status in older adults. Reliability of gait variability has been examined mainly in community dwelling older adults who are likely to fluctuate over time. The purpose of this study was to compare test–retest reliability and determine minimal detectable change (MDC) of spatial and temporal gait variability in younger and older adults. Forty younger (mean age = 26.6 ± 6.0 years) and 46 older adults (mean age = 78.1 ± 6.2 years) were included in the study. Gait characteristics were measured twice, approximately 1 week apart, using a computerized walkway (GaitMat II). Participants completed 4 passes on the GaitMat II at their self-selected walking speed. Test-retest reliability was calculated using Intra-class correlation coefficients (ICCs(2,1)), 95% limits of agreement (95% LoA) in conjunction with Bland-Altman plots, relative limits of agreement (LoA%) and standard error of measurement (SEM). The MDC at 90% and 95% level were also calculated. ICCs of gait variability ranged 0.26–0.65 in younger and 0.28–0.74 in older adults. The LoA% and SEM were consistently higher (i.e. less reliable) for all gait variables in older compared to younger adults except SEM for step width. The MDC was consistently larger for all gait variables in older compared to younger adults except step width. ICCs were of limited utility due to restricted ranges in younger adults. Based on absolute reliability measures and MDC, younger had greater test–retest reliability and smaller MDC of spatial and temporal gait variability compared to older adults.