Abstract Background The impact of hydrothermal flowthrough (FT) pretreatment severity on pretreatment and solubilization performance metrics was evaluated for three milled feedstocks (corn stover, bagasse, and poplar) and two conversion systems (simultaneous saccharification and fermentation using yeast and fungal cellulase, and fermentation by Clostridium thermocellum). Results Compared to batch pretreatment, FT pretreatment consistently resulted in higher XMG recovery, higher removal of non-carbohydrate carbon and higher glucan solubilization by simultaneous saccharification and fermentation (SSF). XMG recovery was above 90% for FT pretreatment below 4.1 severity but decreased at higher severities, particularly for bagasse. Removal of non-carbohydrate carbon during FT pretreatment increased from 65% at low severity to 80% at high severity for corn stover, and from 40% to 70% for bagasse and poplar. Solids obtained by FT pretreatment were amenable to high conversion for all of the feedstocks and conversion systems examined. The optimal time and temperature for FT pretreatment on poplar were found to be 16 min and 210°C. At these conditions, SSF glucan conversion was about 85%, 94% of the XMG was removed, and 62% of the non carbohydrate mass was solubilized. Solubilization of FT-pretreated poplar was compared for C. thermocellum fermentation (10% inoculum), and for yeast-fungal cellulase SSF (5% inoculum, cellulase loading of 5 and 10 FPU/g glucan supplemented with β-glucosidase at 15 and 30 U/g glucan). Under the conditions tested, which featured low solids concentration, C. thermocellum fermentation achieved faster rates and more complete conversion of FT-pretreated poplar than did SSF. Compared to SSF, solubilization by C. thermocellum was 30% higher after 4 days, and was over twice as fast on ball-milled FT-pretreated poplar. Conclusions XMG removal trends were similar between feedstocks whereas glucan conversion trends were significantly different, suggesting that factors in addition to XMG removal impact amenability of glucan to enzymatic attack. Corn stover exhibited higher hydrolysis yields than bagasse or poplar, which could be due to higher removal of non-carbohydrate carbon. XMG in bagasse is more easily degraded than XMG in corn stover and poplar. Conversion of FT-pretreated substrates at low concentration was faster and more complete for C. thermocellum than for SSF.