Abstract Production of a broad spectrum of products from biomass is of key importance for an economically viable and sustainable biorefinery sector. The aim of this study was to determine the total crude protein yield and theoretical extractable true protein of potential biorefinery feedstocks optimized for supplying biomass to biorefineries. Field experiments during 2013–2014 with perennial crops (pure grasses: cocksfoot, festulolium, reed canary, tall fescue, two miscanthus species and two grass-legume mixtures) and annual crops in optimized rotations (winter rye, sugar beet, maize, triticale, hemp and grass-clover) were compared to traditional crops common in Danish agriculture (maize, barley, wheat and triticale). Theoretical extractable true protein was determined according to the Cornell Net Carbohydrate and Protein System, which fractionates the crude protein based on solubilities. The easily extractable fraction of the true protein was denoted as neutral-extractable. Concentration of crude protein was on average 164–191 g kg−1 DM per cut of pure grasses and grass-legume mixtures, with the summer cuts having the lowest values. Pure grasses produced the highest crude protein yield per hectare annually, ranging from 2595 to 3693 kg ha−1 irrespective of the year, of which 920–1640 kg ha−1 was neutral-extractable protein. Whilst the neutral-extractable true protein per hectare of festulolium and tall fescue was superior to those of all other crops, the neutral extractable true protein per hectare of grass-legume mixtures and of winter rye and maize double crop was similar to those of reed canary and cocksfoot. On a mass basis, 34–46% of crude protein in pure grasses was neutral-extractable, depending on the year. The potential extractability of crude protein may be increased by 14–35% if the cell wall-bound protein can be extracted too.