MicroRNAs (miRNAs) are important regulators of gene expression in multicellular organisms. Yet, little is known about their molecular evolution. The 20- to 22-nt long miRNAs are processed in plants from foldbacks that are a few hundred base pairs in size. Often, these foldbacks are embedded in much larger precursor transcripts. To investigate functional constraints on sequence evolution of miRNA precursor genes, we have studied sequence variation in the precursor of miR319a, MIR319a, between species from the Brassicaceae. We compared the genomic context in Arabidopsis thaliana, Arabidopsis halleri, and Capsella rubella, using bacterial artificial chromosome clones, and analyzed precursor sequences obtained by polymerase chain reaction from 13 additional species. Phylogenetic shadowing identifies a conserved motif around the transcription start site, which we demonstrate to be functionally important. We further assessed the functionality of MIR319a orthologs from several Brassicaceae species in A. thaliana. The ortholog from kale (Brassica oleracea var. acephala) was found to be largely inactive, at least partially due to mutations in the miRNA itself, but experimental evidence suggests that loss of miR319a function is compensated by other members of the miR319 family. More broadly, we find that the foldback diverges less rapidly than the remainder of the primary transcript. To understand the molecular evolution of miRNA genes, investigations at different levels of phylogenetic divergence are required.