MicroRNAs (miRNAs) are short endogenous RNA molecules that regulate gene expression at the posttranscriptional level and have been shown to play critical roles during animal development. The identification and comparison of miRNAs in metazoan species are therefore paramount for our understanding of the evolution of body plans. We have characterized 203 miRNAs from the red flour beetle Tribolium castaneum by deep sequencing of small RNA libraries. We can conclude, from a single study, that the Tribolium miRNA set is at least 15% larger than that in the model insect Drosophila melanogaster (despite tens of highthroughput sequencing experiments in the latter). The rate of birth and death of miRNAs is high in insects. Only one-third of the Tribolium miRNA sequences are conserved in D. melanogaster, and at least 18 Tribolium miRNAs are conserved in vertebrates but lost in Drosophila. More than one-fifth of miRNAs that are conserved between Tribolium and Drosophila exhibit changes in the transcription, genomic organization, and processing patterns that lead to predicted functional shifts. For example, 13% of conserved miRNAs exhibit seed shifting, and we describe arm-switching events in 11% of orthologous pairs. These shifts fundamentally change the predicted targets and therefore function of orthologous miRNAs. In general, Tribolium miRNAs are more representative of the insect ancestor than Drosophila miRNAs and are more conserved in vertebrates. © The Author(s) 2010.