AbstractMiRNAs are crucial regulators of gene expression found across both the plant and animal kingdoms. While the number of annotated miRNAs deposited in miRBase has greatly increased in recent years, few studies provided comparative analyses across sets of related species, or investigated the role of miRNAs in the evolution of gene regulation. We generated small RNA libraries across 5 mammalian species (cow, dog, horse, pig and rabbit) from 4 different tissues (brain, heart, kidney and testis). We identified 1676 miRBase and 413 novel miRNAs by manually curating the set of computational predictions obtained from miRCat and miRDeep2. Our dataset spanning five species has enabled us to investigate the molecular mechanisms and selective pressures driving the evolution of miRNAs in mammals. We highlight the important contributions of intronic sequences (366 orthogroups), duplication events (135 orthogroups) and repetitive elements (37 orthogroups) in the emergence of new miRNA loci. We use this framework to estimate the patterns of gains and losses across the phylogeny, and observe high levels of miRNA turnover. Additionally, the identification of lineage-specific losses enables the characterisation of the selective constraints acting on the associated target sites. Compared to the miRBase subset, novel miRNAs tend to be more tissue specific. 20 percent of novel orthogroups are restricted to the brain, and their target repertoires appear to be enriched for neuron activity and differentiation processes. These findings may reflect an important role for young miRNAs in the evolution of brain expression plasticity. Many seed sequences appear to be specific to either the cow or the dog. Analyses on the associated targets highlight the presence of several genes under artificial positive selection, suggesting an involvement of these miRNAs in the domestication process. Altogether, we provide an overview on the evolutionary mechanisms responsible for miRNA turnover in 5 domestic species, and their possible contribution to the evolution of gene regulation.