Abstract Major differences exist between plants and animals both in the extent of microRNA (miRNA)-based gene regulation and the sequence complementarity requirements for miRNA-messenger RNA pairing. Whether these differences affect how these sites evolve at the molecular level is unknown. To determine the extent of sequence variation at miRNAs and their targets in a plant species, we resequenced 16 miRNA families (66 miRNAs in total) and all 52 of the characterized binding sites for these miRNAs in the plant model Arabidopsis (Arabidopsis thaliana), accounting for around 50% of the known miRNAs and binding sites in this species. As has been shown previously in humans, we find that both miRNAs and their target binding sites have very low nucleotide variation and divergence compared to their flanking sequences in Arabidopsis, indicating strong purifying selection on these sites in this species. Sequence data flanking the mature miRNAs, however, exhibit normal levels of polymorphism for the accessions in this study and, in some cases, nonneutral evolution or subtle effects on predicted pre-miRNA secondary structure, suggesting that there is raw material for the differential function of miRNA alleles. Overall, our results show that despite differences in the architecture of miRNA-based regulation, miRNAs and their targets are similarly constrained in both plants and animals.