AbstractBreeding between Saccharomyces species is a useful tool for obtaining improved wine yeast strains, combining fermentative features of parental species. In this work, 25 artificial Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed by spore conjugation. A multi‐locus PCR–restriction fragment length polymorphism (PCR–RFLP) analysis, targeting six nuclear gene markers and the ribosomal region including the 5.8S rRNA gene and the two internal transcribed spacers, showed that the hybrid genome is the result of two chromosome sets, one coming from S. cerevisiae and the other from S. uvarum. Mitochondrial DNA (mtDNA) typing showed uniparental inheritance in all hybrids. Furthermore, sibling hybrids, obtained by repeated crosses between the same parental strains, showed the same mtDNA, suggesting that the mitochondrial transmission is not stochastic or species‐specific, but dependent on the parental strains. Finally four hybrids, two of which with S. cerevisiae mtDNA and two with S. uvarum mtDNA, were subjected to transcriptome analysis. Our results showed that the hybrids bearing S. cerevisiae mtDNA exhibited less expression of genes involved in glycolysis/fermentation pathways and in hexose transport compared to hybrids with S. uvarum mtDNA. Respiration assay confirmed the increased respiratory activity of hybrids with the S. cerevisiae mtDNA genome. These findings suggest that mtDNA type and fermentative : respiratory performances are correlated in S. cerevisiae × S. uvarum hybrids and the mtDNA type is an important trait for constructing new improved hybrids for winemaking. Copyright © 2008 John Wiley & Sons, Ltd.