H5 highly pathogenic avian influenza viruses (HPAIVs) are prevalent in birds, causing infection in over 60 mammalian species worldwide. However, species-specific viral-host response at the single-cell level and within-host evolution upon H5-HPAIV infection are poorly understood. Here, by integrating spatial transcriptomics and single-cell genomics technologies, we present a comprehensive single-cell transcriptomic landscape of 1499?669 cells in the lungs from birds (chickens, ducks, and pigeons) and mammals (mice and pigs) upon H5-HPAIV infection. H5-HPAIV primarily infected avian endothelial cells and mammalian epithelial cells, respectively. Among the single-cell transcriptomic lung landscape, H5-HPAIV infection induced significant inflammation in fibroblasts and the shedding of alveolar epithelial cells of chickens upon infection, while the expression levels of cytokine- and inflammation-related genes slightly decreased in fibroblasts and endothelial cells of infected ducks and pigeons, respectively. However, mice only exhibited significantly antiviral response in macrophages and neutrophils, while antiviral response was elevated in most subtype cells of pigs upon infection. Notably, we identified 19 viral non-synonymous intrahost single nucleotide variants (iSNVs) in infected birds and mammals and assessed their molecular characterizations in vitro. Three of them were identified to increase polymerase activity and viral replication in situ, among which the PB2-E627K mutation was enriched in the alveolar structure of lungs, accompanied by elevated expression of interferon-stimulated genes. Collectively, we elucidated distinct viral replication, host response, and viral adaptation in the lungs across diverse birds and mammals upon H5-HPAIV infection.