High yields are a priority in managing biomass for renewable energy, but the environmental impacts of various feedstocks and production systems should be equally considered. Mixed-species, perennial grasslands enrolled in conservation programs are being considered as a source of biomass for renewable energy. Conservation grasslands are crucial in sustaining native biodiversity throughout the US Upper Midwest, and the effects of biomass harvest on biodiversity are largely unknown. We measured the effect of late-season biomass harvest on plant community composition in conservation grasslands in three regions of Minnesota, USA from 2009 to 2012. Temporal trends in plant species composition within harvested grasslands were compared to unharvested grasslands using mixed effects models. A before-after control-impact approach using effect sizes was applied to focus on pre- and postharvest conditions. Production-scale biomass harvest did not affect plant species richness, species or functional group diversity, nor change the relative abundance of the main plant functional groups. Differences in the relative abundances of plant functional groups were observed across locations; and at some locations, changed through time. The proportion of non-native species remained constant, while the proportion of noxious weeds decreased through time in both harvested and unharvested grasslands at the central location. Ordination revealed patterns in species composition due to location, but not due to harvest treatment. Therefore, habitat and bioenergy characteristics related to grassland plant communities are not expected to change due to short-term or intermittent late-season biomass harvest.