Heterokaryosis is a system in which genetically distinct nuclei coexist within the same cytoplasm. While heterokaryosis dominates the life cycle of many fungal species, the transcriptomic changes associated with the transition from homokaryosis to heterokaryosis is not well understood. Here, we analyse gene expression profiles of homokaryons and heterokaryons from three phylogenetically and reproductively isolated lineages of the filamentous ascomycete . We show that heterokaryons are transcriptionally distinct from homokaryons in the sexual stage of development, but not in the vegetative stage, suggesting that the phenotypic switch to fertility in heterokaryons is associated with major changes in gene expression. Heterokaryon expression is predominantly defined by additive effects of its two nuclear components. Furthermore, allele-specific expression analysis of heterokaryons with varying nuclear ratios show patterns of expression ratios strongly dependent on nuclear ratios in the vegetative stage. By contrast, in the sexual stage, strong deviations of expression ratios indicate a co-regulation of nuclear gene expression in all three lineages. Taken together, our results show two levels of expression control: additive effects suggest a nuclear level of expression, whereas co-regulation of gene expression indicate a heterokaryon level of control.