Natural range of variation (NRV) may be used to establish decision thresholds or action assessment points when ecological thresholds are either unknown or do not exist for attributes of interest in a managed ecosystem. The process for estimating NRV involves identifying spatial and temporal scales that adequately capture the heterogeneity of the ecosystem; compiling data for the attributes of interest via study of historic records, analysis and interpretation of proxy records, modeling, space-for-time substitutions, or analysis of long-term monitoring data; and quantifying the NRV from those data. At least 19 National Park Service (NPS) units in North America’s Great Plains are monitoring plant species richness and evenness as indicators of vegetation integrity in native grasslands, but little information on natural, temporal variability of these indicators is available. In this case study, we use six long-term vegetation monitoring datasets to quantify the temporal variability of these attributes in reference conditions for a variety of Great Plains grassland types, and then illustrate the implications of using different NRVs based on these quantities for setting management decision thresholds. Temporal variability of richness (as measured by the coefficient of variation, CV) is fairly consistent across the wide variety of conditions occurring in Colorado shortgrass prairie to Minnesota tallgrass sand savanna (CV 0.20–0.45) and generally less than that of production at the same sites. Temporal variability of evenness spans a greater range of CV than richness, and it is greater than that of production in some sites but less in other sites. This natural temporal variability may mask undesirable changes in Great Plains grasslands vegetation. Consequently, we suggest that managers consider using a relatively narrow NRV (interquartile range of all richness or evenness values observed in reference conditions) for designating a surveillance threshold, at which greater attention to the situation would be paid, and a broader NRV for designating management thresholds, at which action would be instigated.