Gene expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer 1 . Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profling and matched RNA-sequencing data for three organs (brain, liver and testis) in fve mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the diferent organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifcally counterbalanced global dosage reductions during the evolution of sex chromosomes and the efects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of bufering, some genes evolved faster at the translatome layer—potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is refected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.