Developing an effective and economical biomass pretreatment method is a significant roadblock to meeting the ever growing demand for transportation fuels. Earlier studies with different feedstocks revealed that in the absence of chemicals, neither extrusion nor microwave could be standalone pretreatments. However, there is potential that the advantages of these individual methods can be harnessed in a sequential pretreatment process. Accordingly, switchgrass and big bluestem were extruded and then subject to microwave pretreatment, under optimal conditions that had been separately determined in prior studies. Pretreated biomass was then subject to enzymatic hydrolysis to understand the effectiveness of the sequential pretreatment on sugar recovery and generation of fermentation inhibitors. Statistical analysis confirmed that moisture content, microwave power level, and exposure time (and their interactions) had significant influence on sugar recovery. Sequential pretreatment of switchgrass (25% moisture, 450W and 2.5 min) resulted in a maximum glucose, xylose, and total sugar recovery of 52.6%, 75.5%, and 59.2%, respectively. This was higher by 1.27 and 2.71, 1.21 and 4.60, and 1.25 and 2.87 times compared to extrusion alone and the unpretreated control, respectively. The same sequential pretreatment conditions achieved maximum glucose, xylose, and total sugar recovery of 83.2%, 92.1%, and 68.1%, respectively, for big bluestem. This was 1.14 and 4.1, 1.18 and 2.7, and 1.20 and 3.0 times higher than extrusion alone and the unpretreated control, respectively. This sequential pretreatment process did not aggravate acetic acid formation over levels observed with the individual pretreatments. Furthermore, furfural, HMF, and formic acid were not detected in any of the treatments. Although the sequential pretreatment process enhanced sugar recovery without increasing the levels of potential fermentation inhibitors, the increased energy input for the microwave treatment may not be economical. Published by Elsevier Ltd.