Biomass preprocessing and pretreatment technologies such as size reduction and chemical preconditioning are aimed at reducing the cost of ethanol production from lignocellulosic biomass. Size reduction is an energy-intensive biomass preprocessing unit operation. In this study, switchgrass, wheat straw, and corn stover were chopped in an instrumented knife mill to evaluate size reduction energy and corresponding particle size distribution as determined with a standard forage sieve analyzer. Direct mechanical power inputs were determined using a dedicated data acquisition system for knife mill screen openings from 12.7 to 50.8 mm, rotor speeds between 250 and 500 rpm, and mass feed rates from 1 to 11 kg/min. A speed of 250 rpm gave optimum performance of the mill. Optimum feed rates for 25.4 mm screen and 250 rpm were 7.6, 5.8, and 4.5 kg/min for switchgrass, wheat straw, and corn stover, respectively. Total specific energy (MJ/Mg) was defined as the size reduction energy required to operate the knife mill plus that imparted to the biomass. Effective specific energy was defined as the energy imparted to the biomass. For these conditions, total specific energies were 27.3, 37.9, and 31.9 MJ/Mg and effective specific energies were 10.1, 15.5, and 3.2 MJ/Mg for switchgrass, wheat straw, and corn stover, respectively. These results demonstrated that biomass selection affects the size reduction energy, even for biomass with similar features. Second-order polynomial equations for the total specific energy requirement fitted well (R2 > 0.95) as a function of knife mill screen size, mass feed rate, and speed for biomass materials tested. The Rosin-Rammler equation fitted the cumulative undersize mass of switchgrass, wheat straw, and corn stover chop passed through ASABE sieves with high R2 (>0.983). Knife mill chopping of switchgrass, wheat straw, and corn stover resulted in particle size distributions classified as 'well-graded strongly fine-skewed mesokurtic', 'well-graded fine-skewed mesokurtic', and 'well-graded fine-skewed mesokurtic', respectively, for small knife mill screen sizes (12.7 to 25.4 mm) and distributions classified as 'well-graded fine-skewed mesokurtic', 'well-graded strongly fine-skewed mesokurtic', and 'well-graded fine-skewed mesokurtic', respectively, for the large screen size (50.8 mm). Total and effective specific energy values per unit size reduction of wheat straw were greater compared to those for switchgrass. Corn stover resulted in reduced total and effective specific energy per unit size reduction compared to wheat straw for the same operating conditions, but higher total specific energy per unit size reduction and lesser effective specific energy per unit size reduction compared to switchgrass. Data on minimized total specific energy with corresponding particle spectra will be useful for preparing feed material with a knife mill for subsequent grinding with finer size reduction devices.