Uncertainty is an important factor in ecological assessment, and has important implications for the ecological classification and management of lakes. However, our knowledge of the effects of uncertainty in the assessment of different ecological indicators is limited. Here, we used data from a standardized campaign of aquatic plant surveys, in 28 lakes from 10 European countries, to assess variation in macrophyte metrics across a set of nested spatial scales: countries, lakes, sampling stations, replicate transects, and replicate samples at two depth-zones. Metrics investigated in each transect included taxa richness, maximum depth of colonisation and two indicators of trophic status: Ellenberg’s N and a metric based on phosphorus trophic status. Metrics were found to have a slightly stronger relationship to pressures when they were calculated on abundance data compared to presence/absence data. Eutrophication metrics based on helophytes were found not to be useful in assessing the effects of nutrient pressure. These metrics were also found to vary with the depth of sampling, with shallower taxa representing higher trophic status. This study demonstrates the complex spatial variability in macrophyte communities, the effect of this variability on the metrics, and the implications to water managers, especially in relation to survey design.