Two-step steam pretreatment of softwood on laboratory scale has previously been shown to result in higher yields than one-step steam pretreatment. In this study, these results are verified on a larger scale. In an industrial process filtration and washing of the material between the two pretreatment steps are difficult without release of pressure. A worst case without filtration or washing was thus investigated to determine the influence of poor washing on the yield of sugars and the formation of byproducts. Steam pretreatment with SO2 impregnation was investigated using three different procedures. One-step steam pretreatment was performed at 215 °C for 5 min. Two different kinds of two-step steam pretreatment were performed at 190 °C for 2 min in the first step and at 210 °C for 5 min in the second step. In one case the slurry obtained after the first pretreatment step was separated into a liquid and a solid phase, where the water-insoluble solid material was washed with water and then used for pretreatment in the second step. In the other case of two-step steam pretreatment, neither separation nor washing was performed. The pretreated material was evaluated using both enzymatic hydrolysis and fed-batch simultaneous saccharification and fermentation. Both two-step steam pretreatment process configurations investigated resulted in higher yields of ethanol (300 L/ton) than one-step steam pretreatment (227 L/ton). Separation and washing of the material between the pretreatment steps in the two-step steam pretreatment process did not improve the overall sugar yield, although the formation of sugar degradation products was reduced.