Abstract Only trace amounts of sulfur (tens to thousands of ppm) are present in bio-oils produced from fast pyrolysis of lignocellulosic biomass. However, even such small amounts of sulfur-containing compounds can act as catalytic poisons during bio-oil upgrading. To improve the knowledge of sulfur speciation in bio-oils for process design and development, e.g. by hydrotreatment, comprehensive two-dimensional gas chromatography (GC ×GC) coupled with selective sulfur chemiluminescence detector (SCD) and headspace gas chromatography coupled to a quadrupole mass spectrometry (GC–MS) were combined. This allowed to quantify sulfur-containing compounds present in crude bio-oils produced from different types of biomass (beech wood, miscanthus, and straw) and straw bio-oil after hydrotreatment. Hydrogen sulfide, methanethiol, dimethyl disulfide, and several thiophenes were identified and quantified as the most abundant sulfur compounds. The detailed analysis of the hydrotreated bio-oil prepared at 360 °C and 8 MPa showed that most GC-detectable sulfur was related to hydrogen sulfide not sufficiently removed in the product separator. Used analytical methods brought an unprecedented level of details about bio-oil sulfur speciation and acquired data can help to drive further R&D in bio-oil upgrading.