The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae x Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 A degrees P) and very high gravity wort (25 A degrees P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (ATF1, ATF2A EHT1, EEB1, and BAT1) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the S. cerevisiae- and S. eubayanus-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.