Landscape representations based on land cover databases differ significantly from the real landscape. Using a land cover database with high uncertainty as input for emission inventory analyses can cause propagation of systematic and random errors. The objective of this study was to analyze how different land cover representations introduce systematic errors into the results of regional N2O emission inventories. Surface areas of grassland, ditches, and ditch banks were estimated for two polders in the Dutch fen meadow landscape using five land cover representations: four commonly used databases and a detailed field map, which most closely resembles the real landscape. These estimated surface areas were scaled up to the Dutch western fen meadow landscape. Based on the estimated surface areas agricultural N2O emissions were estimated using different inventory techniques. All four common databases overestimated the grassland area when compared to the field map. This caused a considerable overestimation of agricultural N2O emissions, ranging from 9% for more detailed databases to 11% for the coarsest database. The effect of poor land cover representation was larger for an inventory method based on a process model than for inventory methods based on simple emission factors. Although the effect of errors in land cover representations may be small compared to the effect of uncertainties in emission factors, these effects are systematic (i.e., cause bias) and do not cancel out by spatial upscaling. Moreover, bias in land cover representations can be quantified or reduced by careful selection of the land cover database.