AbstractSustainability of biogas production is strongly dependent on soil‐borne greenhouse gas (GHG) emissions during feedstock cultivation. Maize (Zea mays) is the most common feedstock for biogas production in Europe. Since it is an annual crop requiring high fertilizer input, maize cropping can cause high GHG emissions on sites that, due to their hydrology, have high N2O emission potential. On such sites, cultivation of cup plant (Silphium perfoliatum) as a perennial crop could be a more environmentally friendly alternative offering versatile ecosystem services. To evaluate the possible benefits of perennial cup plant cropping on GHG emissions and nitrogen losses, an incubation study was conducted with intact soil cores from a maize field and a cup plant field. The 15N gas flux method was used to quantify N source‐specific N2 and N2O fluxes. Cumulated N2O emissions and N2+N2O emissions did not differ significantly between maize and cup plant soils, but tended to be higher in maize soil. Soils from both systems exhibited relatively high and similar N2O/(N2+N2O) ratios (N2Oi). N2O emissions originating from sources other than the 15N‐labelled NO3 pool were low, but were the only fluxes exhibiting a significant difference between the maize and cup plant soils. Missing differences in fluxes derived from the 15N pool indicate that under the experimental conditions with high moisture and level, and without plants, the cropping system had little effect on N fluxes related to denitrification. Lower soil pH and higher bulk density in the cup plant soil are likely to have reduced the mitigation potential of perennial biomass cropping.