Chickens are highly susceptible to H5N1 highly pathogenic avian influenza virus (HPAIV) while ducks are not. In this work, we analysed the microRNA expression in the spleen, thymus and bursa of Fabricius of chickens and ducks with and without H5N1 HPAIV infection via high-throughput sequencing. Firstly, we annotated duck microRNAs and compared microRNA repertoires of chickens and ducks. Secondly, in the homologous immune organs of specific-pathogen-free (SPF) chickens and ducks, the microRNA expression patterns showed substantial divergence. Thirdly, we revealed that there was larger divergence in microRNA expression in immune organs of HPAIV-infected chickens than in those of HPAIV-infected ducks. Taken together, our results would be valuable in elucidating the roles of microRNAs in the divergent immunity of chickens and ducks against H5N1 HPAIV.