Hydrolysate pretreatment (HP) uses hot water pre-hydrolysis liquor (HWPL) as partial or full pretreatment medium for biomass. Pentosan dissolution during Eucalyptus HP was studied under holding times between 0 min and 160 min, holding temperatures between 150 °C and 190 °C, a hot water pre-hydrolysate ratio (HWPR) from 20% to 100%, and a fixed liquid to wood ratio of 1:6. Both the pentosan removals in the hydrolysate pretreated solid (HPS) and the hydrolysate pretreatment liquor (HPL) pento-saccharides contents were determined and compared with those of hot water pre-hydrolysis (HWP). When compared to HWP, the HP enhanced pentosan removal from the solid phase, and enriched the saccharides or promoted the in-situ conversion of saccharides into other chemicals in the liquid phase. Pentosan removal in the HPS increased when holding time and temperature were increased. Increasing holding time first increased the pento-saccharide content in the HPL, and then decreased it after reaching the maximum. Elevating the holding temperature increased the pento-saccharide content in the HPL, except for arabino-oligosaccharide. Different HWPR had varying influences on pentosan removal in the HPS and on the saccharides concentration in the HPL. When controlled, HP positively influenced hemicellulose removal from biomass, and increased utilization value of the liquid phase obtained post pretreatment.