Analytical pyrolysis combined with gas chromatography/mass spectrometry (Py-GC/MS) was used to analyze chemical composition of non-hydrolyzed residues (LHRs) obtained by three methods of bioethanol production: softwood acid hydrolysis (AH), separate enzymatic hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF). Complementary techniques, such as EPR- and FTIR-spectroscopy, and routine chemical analysis procedures were used for this study as well. The Py-GC/MS analysis of the LHRs has shown a higher efficiency of carbohydrates hydrolysis upon SSF process in comparison with SHF and AH processes. Comparison of chemical analysis results and data obtained by Py-GC/MS of LHRs brought the direct evidence of incorporation of carbohydrates-derived fragments into the lignin matrix and formation of so-called pseudo-lignin upon different stages of softwood processing. Modifications of lignin component of LHRs on various stages of the process of bioethanol production, such as oxidation and condensation reactions, cleavage of ether bonds and destruction of side propane chain, were revealed using Py-GC/MS.