Chickens are susceptible to the highly pathogenic H5N1 strain of avian influenza virus (HPAIV), whereas ducks are not. Here, we used high-throughput sequencing to analyse the microRNA expression in the spleen, thymus and bursa of Fabricius of H5N1-HPAIV-infected and non-infected chickens and ducks. We annotated the genomic positions of duck microRNAs and we compared the microRNA repertoires of chickens and ducks. Our results showed that the microRNA expression patterns in the homologous immune organs of specific-pathogen-free (SPF) chickens and ducks diverge substantially. Moreover, there was larger divergence between the microRNA expression patterns in immune organs of HPAIV-infected chickens than HPAIV-infected ducks. Together, our results might help to elucidate the roles of microRNAs in the divergent immunity of chickens and ducks against H5N1 HPAIV.