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Whole genome comparison of two Starmerella bacillaris strains with other wine yeasts uncovers genes involved in modulating important winemaking traits

&NA; Starmerella bacillaris is an osmotolerant yeast with interesting winemaking traits such as low‐ethanol and high‐glycerol production, previously considered as wine spoilage and recently proposed to improve the sensory quality of wine. This is the first work performing a whole‐genome analysis of the variants identified by comparing two S. bacillaris strains (PAS13 and FRI751). Additionally, an extensive search for orthologous genes against Saccharomyces and non‐Saccharomyces yeasts produced a detailed reconstruction of the pan‐genome for yeast species used in winemaking. Starmerella bacillaris PAS13 was able to produce 36% more glycerol than S. bacillaris FRI751 without increasing ethanol level over 5% (v/v). Orthologous genes revealed new insights in the response to osmotic stress determined by the mitogen‐activated protein kinase (MAPK) from S. bacillaris strains. The comparison between the two S. bacillaris genomes revealed 33 771 high‐quality variants that were ranked considering their predicted impact on gene functions. Furthermore, analysis of structural variations in the genome revealed five translocations. The absence of some transcriptional factors involved in the regulation of GPD (glycerol‐3‐phosphate dehydrogenase), like the protein kinases YpK1p and YpK2p, and the identification of a tandem duplication increasing the GPP1 (glycerol‐3‐phosphate phosphatase) gene copy number suggest a remarkably different regulation of the glycerol pathway for S. bacillaris in comparison to S. cerevisiae.