Beer production generates brewer's spent grain as a byproduct. This biomass contains lignin, cellulose, and hemicellulose, and can be used for the production of second‐generation (2G) ethanol and xylitol. For this to be economically viable, fermentable fractions must be used, allowing the conversion of cellulose and hemicellulose to ethanol. This study evaluated the fermentation of hemicellulose liquor from brewer's spent grain (HLBS) in aerobic and oxygen‐ limited conditions using Scheffersomyces stipitis and Pachysolen tannophilus. Results were compared with data obtained in a complex medium (YPX) and a minimal medium (MMX). In aerobic conditions, for S. stipitis in the HLBS medium, μ max = 0.12 h−1, Y ethanol/xylose = 0.14 and selectivity for ethanol over xylitol (S ethanol/xylitol) 3.4 gethanol g−1 xylitol were obtained. Under oxygen‐limited conditions, Y ethanol/xylose = 0.22 and S ethanol/xylitol = 1.17 gethanol g−1 xylitol were obtained. For P. tannophilus, under aerobic conditions, the HLBS medium gave μ max = 0.08 h−1, and Y ethanol/xylose = 0.005, with selectivity for ethanol over xylitol (S ethanol/xylitol) of 0.12 gethanol g−1 xylitol. In oxygen‐limited conditions, Y ethanol/xylose = 0.09 and S ethanol/xylitol = 0.20 gethanol g−1 xylitol were obtained. This is because the metabolism of these aerobic yeasts directs the carbon flow to cell growth, while with oxygen limitation it is directed to metabolite production. The HLBS medium was promising for production in the context of a biorefinery. When comparing the two yeasts, S. stipitis presented better ethanol production and P. tannophilus better xylitol production under the conditions that were studied. © 2019 Society of Chemical Industry and John Wiley & Sons, Ltd