The recent introduction and spread of HPAI A(H5N1) clade 2.3.4.4b viruses to U.S. dairy cattle demonstrate their ability to adapt to new mammalian hosts. Although multiple A(H5N1) genotypes have caused human infections, only two clade 2.3.4.4b genotypes (B3.13 and D1.1) have been detected in cattle, suggesting clade- or genotype-specific tropism. Understanding how these viruses replicate in human airway and bovine mammary epithelial cells is important for assessing viral evolution, mammalian adaptation, and associated public health risks. Here, we characterized replication kinetics and immune gene expression in human bronchial epithelial (Calu-3) cells and bovine mammary epithelial (MAC-T) cells infected with A(H5N1) clade 2.3.4.4b human isolates representing the B3.13, D1.1, and B3.2 genotypes, an A(H5N1) clade 1 virus, and an A(H1N1)pdm09 strain. All viruses replicated efficiently in Calu-3 cells, although replication was delayed at 33°C compared to 37°C. Clade 2.3.4.4b viruses induced moderate expression of type I interferon and proinflammatory response in Calu-3 cells, but at lower levels than the A(H1N1)pdm09 and A(H5N1) clade 1 strains. Replication and immune activation varied markedly among clade 2.3.4.4b genotypes in MAC-T cells. B3.13 viruses achieved higher titers and triggered stronger induction of innate immune response genes than other A(H5N1) genotypes or clades. These findings indicate genotype- and cell type–specific differences in replication and host responses, with B3.13 showing enhanced tropism in bovine mammary cells, consistent with adaptation that may elevate zoonotic risk. Our results highlight the value of comparative studies across host cell types and diverse viral isolates to inform risk assessments for emerging influenza viruses.