Background: MicroRNAs have been identified as crucial regulators in both animals and plants.Here we report on a comprehensive comparative study of all known miRNA families in animals.We expand the MicroRNA Registry 6.0 by more than 1000 new homologs of miRNA precursorswhose expression has been verified in at least one species. Using this uniform data basis we analyzetheir evolutionary history in terms of individual gene phylogenies and in terms of preservation ofgenomic nearness across species. This allows us to reliably identify microRNA clusters that arederived from a common transcript. Results: We identify three episodes of microRNA innovation that correspond to majordevelopmental innovations: A class of about 20 miRNAs is common to protostomes anddeuterostomes and might be related to the advent of bilaterians. A second large wave ofinnovations maps to the branch leading to the vertebrates. The third significant outburst of miRNAinnovation coincides with placental (eutherian) mammals. In addition, we observe the expectedexpansion of the microRNA inventory due to genome duplications in early vertebrates and in anancestral teleost. The non-local duplications in the vertebrate ancestor are predated by local(tandem) duplications leading to the formation of about a dozen ancient microRNA clusters. Conclusion: Our results suggest that microRNA innovation is an ongoing process. Majorexpansions of the metazoan miRNA repertoire coincide with the advent of bilaterians, vertebrates,and (placental) mammals.