AbstractAmid known microbial bioethanol producers, the yeast Scheffersomyces (Pichia) stipitis is particularly promising in terms of alcoholic fermentation of both glucose and xylose, the main constituents of lignocellulosic biomass hydrolysates. However, the ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a feasible industrial technology; therefore, the construction of more efficient S. stipitis ethanol producers is of great significance. The aim of this study was to isolate the insertional mutants of S. stipitis with altered ethanol production from glucose and xylose and to identify the disrupted gene(s). Mutants obtained by random insertional mutagenesis were screened for their growth abilities on solid media with different sugars and for resistance to 3‐bromopyruvate. Of more than 1,300 screened mutants, 17 were identified to have significantly changed ethanol yields during the fermentation. In one of the best fermenting strains (strain 4.6), insertion was found to occur within the ORF of a homolog to the Saccharomyces cerevisiae gene HEM25 (YDL119C), encoding a mitochondrial glycine transporter required for heme synthesis. The role of HEM25 in heme accumulation, respiration, and alcoholic fermentation in the yeast S. stipitis was studied using strain 4.6, the complementation strain Comp—a derivative from the 4.6 strain with expression of the WT HEM25 allele and the deletion strain hem25Δ. As hem25Δ produced lower amounts of ethanol than strain 4.6, we assume that the phenotype of strain 4.6 may be caused not only by HEM25 disruption but additionally by some point mutation.