Monitoring herd lameness prevalence has utility for dairy producers and veterinarians in their efforts to reduce lameness, for animal welfare assessment programs, and for researchers. Locomotion scoring is a method used to quantify lameness and calculate prevalence. Because of the time necessary to locomotion score each cow in large dairy herds, a sampling strategy to determine herd lameness prevalence that allows scoring of fewer cows would be useful. Such a sampling strategy must be validated for accuracy compared with the lameness prevalence when all cows in a herd are locomotion scored. The purpose of this study was to assess 3 previously suggested methods of estimating lameness prevalence by strategic sampling of dairy herds. Sampling strategies tested included (1) sampling a calculated number of cows in the middle third of the milking parlor exit order for each pen, (2) sampling a calculated number of cows weighted across pens and distributed evenly within each pen, and (3) sampling all cows in the high production, low production, and hospital pens. Lactating cows on 5 dairy farms in Washington and Oregon (n=4,422) were locomotion scored using a 5-point scale to determine herd-level lameness prevalence (percentage with locomotion score ≥3). Milking parlor exit order, order in headlocks at the feed bunk within each pen, and breed were recorded for each cow. The number of days in lactation, milk production, and parity were collected from farm computer records. Pen grouping strategy for each farm was obtained by interview with farm management. Sampling strategies were modeled using the locomotion score data set for each herd. Estimates of lameness prevalence obtained from the milking parlor exit order sample and the sample distributed across pens were within 5 percentage points of the whole herd prevalence. The third strategy estimated the lameness prevalence within 5 percentage points on 4 farms, but overestimated prevalence on 1 farm. Pen-level prevalence obtained by locomotion score of all cows in the pen was variable and not reliably predictive of herd-level prevalence. Cows of Holstein breed, parity >1, and exiting the milking parlor in the last 20% of the pen had greater odds of lameness compared with other breeds, parities, and milking parlor exit order groups in a multivariate analysis. This study indicates that the sampling strategies using the middle of milking parlor exit order and a calculated sample distributed across the herd may be used to obtain an estimate of herd lameness prevalence.