Abstract Background Understanding factors that govern lignocellulosic biomass recalcitrance is a prerequisite for designing efficient 2nd generation biorefining processes. However, the reasons and mechanisms responsible for quantitative differences in enzymatic digestibility of various biomass feedstocks in response to hydrothermal pretreatment at different severities are still not sufficiently understood. Results Potentially important lignocellulosic feedstocks for biorefining, corn stover (Zea mays subsp. mays L.), stalks of Miscanthus × giganteus, and wheat straw (Triticum aestivum L.) were systematically hydrothermally pretreated; each at three different severities of 3.65, 3.83, and 3.97, respectively, and the enzymatic digestibility was assessed. Pretreated samples of Miscanthus × giganteus stalks were the least digestible among the biomass feedstocks producing ~24 to 66.6% lower glucose yields than the other feedstocks depending on pretreatment severity and enzyme dosage. Bulk biomass composition analyses, 2D nuclear magnetic resonance, and comprehensive microarray polymer profiling were not able to explain the observed differences in recalcitrance among the pretreated feedstocks. However, methods characterizing physical and chemical features of the biomass surfaces, specifically contact angle measurements (wettability) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (surface biopolymer composition) produced data correlating pretreatment severity and enzymatic digestibility, and they also revealed differences that correlated to enzymatic glucose yield responses among the three different biomass types. Conclusion The study revealed that to a large extent, factors related to physico-chemical surface properties, namely surface wettability as assessed by contact angle measurements and surface content of hemicellulose, lignin, and wax as assessed by ATR-FTIR rather than bulk biomass chemical composition correlated to the recalcitrance of the tested biomass types. The data provide new insight into how hydrothermal pretreatment severity affects surface properties of key Poaceae lignocellulosic biomass and may help design new approaches to overcome biomass recalcitrance.