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Changes in livestock production systems have led to land-use changes and abandonment, especially of semi-natural grassland in agriculturally less favoured regions. The generation of energy from biomass of extensive, high-diversity grasslands can be an alternative to their abandonment, and anaerobic digestion is one possible method for converting grassland biomass into energy. However, little is known about the effects of species richness (SR) and functional groups on chemical constituents relevant for anaerobic digestion and the resulting energy potential. In this study, changes in the herbage chemical constituents that are relevant for forage quality were studied along a well-defined diversity gradient (one to sixty species) and across different combinations of functional groups (legumes, small herbs, tall herbs and grasses). Substrate-specific methane yield (CH4 sub) was estimated through the concentrations of forage-quality parameters such as crude fibre (CF), crude protein (CP), crude lipid, nitrogen-free extract and their documented digestibility values, as well as the respective methane yields. Results show that with increasing SR, the CF increased and CP decreased, even though these effects could not be fully disentangled from the presence of grasses. These trends led to a negative effect of SR on CH4 sub, while the area-specific methane yield (CH4 area = CH4 sub x biomass yield) increased due to a strong increase in biomass with increasing SR. The CH4 sub was increased when legumes were present, and it declined with the presence of grasses. Generally, CH4 sub and CH4 area varied between functional-group monocultures and all functional-group mixtures.