Abstract Background Mixtures of prairie species (mixed prairie species; MPS) have been proposed to offer important advantages as a feedstock for sustainable production of fuels and chemicals. Therefore, understanding the performance in hydrothermal pretreatment and enzymatic hydrolysis of select species harvested from a mixed prairie is valuable in selecting these components for such applications. This study examined composition and sugar release from the most abundant components of a plot of MPS: a C3 grass (Poa pratensis), a C4 grass (Schizachyrium scoparium), and a legume (Lupinus perennis). Results from this study provide a platform to evaluate differences between grass and leguminous species, and the factors controlling their recalcitrance to pretreatment and enzymatic hydrolysis. Results Significant differences were found between the grass and leguminous species, and between the individual anatomical components that influence the recalcitrance of MPS. We found that both grasses contained higher levels of sugars than did the legume, and also exhibited higher sugar yields as a percentage of the maximum possible from combined pretreatment and enzymatic hydrolysis. Furthermore, particle size, acid-insoluble residue (AcIR), and xylose removal were not found to have a direct significant effect on glucan digestibility for any of the species tested, whereas anatomical composition was a key factor in both grass and legume recalcitrance, with the stems consistently exhibiting higher recalcitrance than the other anatomical fractions. Conclusions The prairie species tested in this study responded well to hydrothermal pretreatment and enzymatic saccharification. Information from this study supports recommendations as to which plant types and species are more desirable for biological conversion in a mixture of prairie species, in addition to identifying fractions of the plants that would most benefit from genetic modification or targeted growth.