A considerable expansion of biogas production in Germany, paralleled by a strong increase in maize acreage, has caused growing concern that greenhouse gas (GHG) emissions during crop substrate production might counteract the GHG emission saving potential. Based on a 2-year field trial, a GHG balance was conducted to evaluate the mitigation potential of regionally adapted cropping systems (continuous maize, maize-wheat-Italian ryegrass, perennial ryegrass ley), depending on nitrogen (N) level and N type. Considering the whole production chain, all cropping systems investigated contributed to the mitigation of GHG emissions (6·7–13·3 t CO2 eq/ha), with continuous maize revealing a carbon dioxide (CO2) saving potential of 55–61% compared with a fossil energy mix reference system. The current sustainability thresholds in terms of CO2 savings set by the EU Renewable Energy Directive could be met by all cropping systems (48–76%). Emissions from crop production had the largest impact on the mitigation effect (⩾50%) unless the biogas residue storage was not covered. The comparison of N fertilizer types showed less pronounced differences in GHG mitigation potential, whereas considerable site effects were observed.