The emergence of the panzootic clade 2.3.4.4b highly pathogenic H5N1 avian influenza in 2020 marked a major expansion in the host range of influenza A viruses (IAVs), raising concerns about further cross-species transmission events and zoonotic spillover. The introduction of 2.3.4.4b viruses into US dairy herds has resulted in widespread circulation, accompanied by reduced milk yield, mastitis and high viral loads in milk. Notably, virus circulation in dairy cattle represents a novel route for mammalian adaptation and transmission that has already led to more than 40 human cases in the USA since 2024. As haemagglutinin (HA) is the primary determinant of viral host range and tissue tropism, we investigated whether viruses containing the glycoproteins of avian H5N1 subclade 2.3.4.4b viruses could infect mammary tissue from Aberdeen Angus, Holstein Friesian and Limousin cattle. Using mammary gland explants, we inoculated tissues with attenuated reassortant viruses expressing the HA and neuraminidase (NA) of three 2.3.4.4b viruses that predated the emergence of H5N1 in the US cattle: A/chicken/England/053052/2021 (AIV07), A/chicken/Scotland/054477/2021 (AIV09) and A/chicken/England/085598/2022 (AIV48). Detection of viral NP antigen by immunohistochemistry in explants from both the teat and gland cistern for all three breeds following inoculation with AIV09 and AIV48 indicates that mammary tissue from each of the three tested cattle breeds is susceptible to infection by viruses bearing H5N1 glycoproteins. Lectin staining showed expression of both α2,3- and α2,6-linked sialic acids in the mammary tissue of all donors, indicating that all three breeds have the potential to support infection with both avian- and mammalian-adapted IAVs. Together, these findings demonstrate that the mammary glands from both beef and dairy cattle breeds are susceptible to infection with viruses bearing avian-origin H5N1 glycoproteins and suggest that bovine mammary tissue may provide an environment in which diverse IAVs could co-infect the same tissue. This possibility underscores the need for further investigation and the inclusion of both beef and dairy cattle in ongoing H5N1 surveillance and risk-assessment