Influenza A viruses (IAVs) have recently expanded their host range to bovine species, raising critical questions about tissue tropism, viral replication capacity, and impacts on dairy production and zoonotic risk. Extensive shedding of highly pathogenic avian influenza virus (HPAIV) H5N1 in milk from naturally infected cattle in the USA highlights the bovine mammary gland as a relevant site of viral replication. To investigate these dynamics, we established and characterized bovine mammary gland organoids (MGOs) derived from primary epithelial cells and maintained in chemically defined media. This efficient, scalable model reconstructs structural and functional hallmarks of the bovine udder, including luminal–basal polarity, lineage-specific cytokeratin patterns, and lactation-associated differentiation. We then used this system to study low and highly pathogenic avian, as well as swine and human IAVs. Viruses from diverse host species productively infected MGOs, largely independent of culturing conditions. Mammalian origin H1N1 strains matched bovine H5N1 in efficiency, but low pathogenic avian H9N2 showed restricted replication, indicating subtype-specific differences in mammary tropism. This suitability study develops a MGO platform that provides a tractable basis for detailed molecular investigation of mammary infection biology and influenza pathogenesis in cattle, to inform strategies that protect both animal and public health.