Whole genome duplication (WGD) has been a major evolutionary driver of increased genomic complexity in vertebrates. One such event occurred in the salmonid family ∼80 million years ago (Ss4R) giving rise to a plethora of structural and regulatory duplicate-driven divergence, making salmonids an exemplary system to investigate the evolutionary consequences of WGD. Here, we present a draft genome of European grayling (Thymallus thymallus), and use this in a comparative framework to study evolution of gene regulation following WGD. Among the Ss4R duplicates identified in European grayling and Atlantic salmon, one third reflect non-neutral tissue expression evolution, with strong purifying selection, maintained over ∼50 million years. Of these, 84% reflect conserved tissue regulation under strong selective constraints and are involved in brain and neural-related functions, as well as higher-order protein-protein interactions. In contrast, 16% of the duplicates have evolved regulatory divergence in a common ancestor, suggestive of adaptive divergence following WGD. These candidates for adaptive expression divergence have elevated rates of protein coding-and promoter sequence evolution, and are enriched for immune-and metabolism ontology terms. Lastly, species-specific duplicate divergence points towards underlying differences in adaptive pressures on expression regulation in the non-anadromous grayling and anadromous Atlantic salmon. Our findings enhance our understanding of the role of WGD in genome evolution and highlights cases of functional divergence of Ss4R duplicates, possibly related to a niche shift in early salmonid evolution.